Functional assays that use the T1R2 taste receptor to identify potential taste modulators

ABSTRACT

Functional assays that use hT1R2 receptor polypeptides to screen for taste modulatory compounds are provided. In preferred embodiments these assays will use cells that stably or transiently express an hT1R2 polypeptide and which further express a suitable protein, e.g. G α15 , G α16  or gustducin. Such functional assays will screen for the effect of compound on T1R2 activity by detecting changes in cell voltage, intracellular ions, phosphorylation, second messenger levels and the like. Compounds identified in such assays are potentially useful as taste modulators. In preferred embodiments, the effect of selected compounds is evaluated further in human taste tests.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/035,045, filed Jan.3, 2002 now U.S. Pat. No. 7,241,880 which application claims benefit ofpriority to U.S. Ser. No. 60/259,227 filed on Jan. 3, 2001 and U.S. Ser.No. 60/284,547, filed on Apr. 19, 2001, both of which are incorporatedby reference in their entirety herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to newly identified mammalian chemosensory Gprotein-coupled receptors, to family of such receptors, and to the genesand cDNA encoding said receptors. More particularly, the inventionrelates to newly identified mammalian chemosensory G protein-coupledreceptors active in taste signaling, to a family of such receptors, tothe genes and cDNA encoding said receptors, and to methods of using suchreceptors, genes, and cDNA in the analysis and discovery of tastemodulators. The invention provides in particular a DNA sequence encodinga novel human taste receptor identified infra as T1R2 and thecorresponding receptor polypeptide.

2. Description of the Related Art

The taste system provides sensory information about the chemicalcomposition of the external world. Mammals are believed to have at leastfive basic taste modalities: sweet, bitter, sour, salty, and umami. See,e.g., Kawamura et al., Introduction to Umami: A Basic Taste (1987);Kinnamon et al., Ann. Rev. Physiol., 54:715-31 (1992); Lindemann,Physiol. Rev., 76:718-66 (1996); Stewart et al., Am. J. Physiol.,272:1-26(1997). Each taste modality is thought to be mediated by adistinct protein receptor or receptors that are expressed in tastereceptor cells on the surface of the tongue (Lindemann, Physol. Rev.76:718-716 (1996)). The taste receptors that recognize bitter, sweet,and umami taste stimuli belong to the G-protein-coupled receptor (GPCR)superfamily (Hoon et al., Cell 96:451 (1999); Adler et al., Cell 100:693(2000)). (Other taste modalities are believed to be mediated by ionchannels.)

G protein-coupled receptors mediate many other physiological functions,such as endocrine function, exocrine function, heart rate, lipolysis,and carbohydrate metabolism. The biochemical analysis and molecularcloning of a number of such receptors has revealed many basic principlesregarding the function of these receptors. For example, U.S. Pat. No.5,691,188 describes how upon a ligand binding to a GPCR, the receptorundergoes a conformational change leading to activation of aheterotrimeric G protein by promoting the displacement of bound GDP byGTP on the surface of the Ga subunit and subsequent dissociation of theGa subunit from the Gb and Gg subunits. The free Ga subunits and Gbgcomplexes activate downstream elements of a variety of signaltransduction pathways.

Complete or partial sequences of numerous human and other eukaryoticchemosensory receptors are currently known. See, e.g., Pilpel, Y. andLancet, D., Protein Science, 8:969-977 (1999); Mombaerts, P., Annu. Rev.Neurosci., 22:487-50 (1999). See also, EP0867508A2, U.S. Pat. No.5,874,243, WO 92/17585, WO 95/18140, WO 97/17444, WO 99/67282. Becauseof the complexity of ligand-receptor interactions, and more particularlytaste stimulus-receptor interactions, information about ligand-receptorrecognition is lacking.

The identification and characterization of the GPCRs that function assweet and umami taste receptors could allow for new methods of discoveryof new taste stimuli. For example, the availability of receptors couldpermit the screening for receptor modulators. Such compounds wouldmodulate taste and could be useful in the food industry to improve thetaste of a variety of consumer products; e.g., improving thepalatability of low-calorie beverages through the development of newartificial sweeteners.

In part, the present invention addresses the need for betterunderstanding of the interactions between chemosensory receptors andchemical stimuli. The present invention also provides, among otherthings, novel chemosensory receptors, and methods for utilizing suchreceptors, and the genes a cDNAs encoding such receptors, to identifymolecules that can be used to modulate chemosensory transduction, suchas taste sensation.

SUMMARY OF THE INVENTION

The invention relates to a new family of G protein-coupled receptors,and to the genes and cDNAs encoding said receptors. The receptors arethought to be primarily involved in sweet taste transduction, but can beinvolved in transducing signals from other taste modalities as well.

The invention provides methods for representing the perception of tasteand/or for predicting the perception of taste in a mammal, including ina human. Preferably, such methods may be performed by using thereceptors and genes encoding said receptors disclosed herein.

Toward that end, it is an object of the invention to provide a newfamily of mammalian G protein-coupled receptors, herein referred to asT1Rs, active in taste perception. It is another object of the inventionto provide fragments and variants of such T1Rs that retain tastestimulus-binding activity.

It is yet another object of the invention to provide nucleic acidsequences or molecules that encode such T1Rs, fragments, or variantsthereof.

It is still another object of the invention to provide expressionvectors which include nucleic acid sequences that encode such T1Rs, orfragments or variants thereof, which are operably linked to at least oneregulatory sequence such as a promoter, enhancer, or other sequenceinvolved in positive or negative gene transcription and/or translation.

It is still another object of the invention to provide human ornon-human cells that functionally express at least one of such T1Rs, orfragments or variants thereof.

It is still another object of the invention to provide T1R fusionproteins or polypeptides which include at least a fragment of at leastone of such T1Rs.

It is another object of the invention to provide an isolated nucleicacid molecule encoding a T1R polypeptide comprising a nucleic acidsequence that is at least 50%, preferably 75%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of: SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, andconservatively modified variants thereof.

It is a further object of the invention to provide an isolated nucleicacid molecule comprising a nucleic acid sequence that encodes apolypeptide having an amino acid sequence at least 35 to 50%, andpreferably 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toan amino acid sequence selected from the group consisting of: SEQ IDNOS: 4, 10, 12, 14, 17, 21, and conservatively modified variantsthereof, wherein the fragment is at least 20, preferably 40, 60, 80,100, 150, 200, or 250 amino acids in length. Optionally, the fragmentcan be an antigenic fragment which binds to an anti-T1R antibody.

It is still a further object of the invention to provide an isolatedpolypeptide comprising a variant of said fragment, wherein there is avariation in at most 10, preferably 5, 4, 3, 2, or 1 amino acidresidues.

It is still another object of the invention to provide agonists orantagonists of such T1Rs, or fragments or variants thereof.

It is yet another object of the invention to provide methods forrepresenting the perception of taste and/or for predicting theperception of taste in a mammal, including in a human. Preferably, suchmethods may be performed by using the T1Rs, or fragments or variantsthereof, and genes encoding such T1Rs, or fragments or variants thereof,disclosed herein.

It is yet another object of the invention to provide novel molecules orcombinations of molecules which elicit a predetermined taste perceptionin a mammal. Such molecules or compositions can be generated bydetermining a value of taste perception in a mammal for a known moleculeor combinations of molecules; determining a value of taste perception ina mammal for one or more unknown molecules or combinations of molecules;comparing the value of taste perception in a mammal for one or moreunknown compositions to the value of taste perception in a mammal forone or more known compositions; selecting a molecule or combination ofmolecules that elicits a predetermined taste perception in a mammal; andcombining two or more unknown molecules or combinations of molecules toform a molecule or combination of molecules that elicits a predeterminedtaste perception in a mammal. The combining step yields a singlemolecule or a combination of molecules that elicits a predeterminedtaste perception in a mammal.

It is still a further object of the invention to provide a method ofscreening one or more compounds for the presence of a taste detectableby a mammal, comprising: a step of contacting said one or more compoundswith at least one of the disclosed T1Rs, fragments or variants thereof,preferably wherein the mammal is a human.

It is another object of the invention to provided a method forsimulating a taste, comprising the steps of: for each of a plurality ofT1Rs, or fragments of variants thereof disclosed herein, preferablyhuman T1Rs, ascertaining the extent to which the T1R interacts with thetaste stimulus; and combining a plurality of compounds, each having apreviously ascertained interaction with one or more of the T1Rs, inamounts that together provide a receptor-stimulation profile that mimicsthe profile for the taste. Interaction of a taste stimulus with a T1Rcan be determined using any of the binding or reporter assays describedherein. The plurality of compounds may then be combined to form amixture. If desired, one or more of the plurality of the compounds canbe combined covalently. The combined compounds substantially stimulateat least 50%, 60%, 70%, 75%, 80% or 90% or all of the receptors that aresubstantially stimulated by the taste stimulus.

In yet another aspect of the invention, a method is provided wherein aplurality of standard compounds are tested against a plurality of T1Rs,or fragments or variants thereof, to ascertain the extent to which theT1Rs each interact with each standard compound, thereby generating areceptor stimulation profile for each standard compound. These receptorstimulation profiles may then be stored in a relational database on adata storage medium. The method may further comprise providing a desiredreceptor-stimulation profile for a taste; comparing the desired receptorstimulation profile to the relational database; and ascertaining one ormore combinations of standard compounds that most closely match thedesired receptor-stimulation profile. The method may further comprisecombining standard compounds in one or more of the ascertainedcombinations to simulate the taste.

It is a further object of the invention to provide a method forrepresenting taste perception of a particular taste stimulus in amammal, comprising the steps of: providing values X₁ to X_(n)representative of the quantitative stimulation of each of n T1Rs of saidvertebrate, where n is greater than or equal to 2; and generating fromsaid values a quantitative representation of taste perception. The T1Rsmay be an taste receptor disclosed herein, or fragments or variantsthereof, the representation may constitutes a point or a volume inn-dimensional space, may constitutes a graph or a spectrum, and mayconstitutes a matrix of quantitative representations. Also, theproviding step may comprise contacting a plurality ofrecombinantly-produced T1Rs, or fragments or variants thereof, with atest composition and quantitatively measuring the interaction of saidcomposition with said receptors.

It is yet another object of the invention to provide a method forpredicting the taste perception in a mammal generated by one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal, comprising the steps of: providing values X₁ to X_(n)representative of the quantitative stimulation of each of n T1Rs of saidvertebrate, where n is greater than or equal to 2; for one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal, providing values X₁ to X_(n) representative of thequantitative stimulation of each of n T1Rs of said vertebrate, where nis greater than or equal to 2; for one or more molecules or combinationsof molecules yielding unknown taste perception in a mammal; andgenerating from said values a quantitative representation of tasteperception in a mammal for the one or more molecules or combinations ofmolecules yielding unknown taste perception in a mammal, and predictingthe taste perception in a mammal generated by one or more molecules orcombinations of molecules yielding unknown taste perception in a mammalby comparing the quantitative representation of taste perception in amammal for the one or more molecules or combinations of moleculesyielding unknown taste perception in a mammal to the quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal. The T1Rs used in this method may include a taste receptor,or fragment or variant thereof, disclosed herein.

DETAILED DESCRIPTION OF THE FIGURES

FIGS. 1 a-1 c present functional data for the human T1R2/T1R3 receptor.Intracellular calcium responses of HEK cells stably expressing Gα15 thatare transiently transfected with human T1R2, T1R3, and T1R2/T1R3 tovarious concentrations of sucrose are shown in FIG. 1 a. Each panelcorresponds to approximately 1000 confluent, transfected, andcalcium-dye-loaded cells. Inhibition of T1R2/T1R3 activity by thesweet-taste inhibitor gurmarin is shown in FIG. 1 b. T1R2/T1R3 doseresponses to four sweeteners and correlated psychophysical detectionthresholds (X-axis circles) are shown in FIG. 1 c.

FIG. 2 presents functional data for the rat T1R2/T1R3 receptor. HumanT1R2/T1R3 and rat T1R2/T1R3 (as well as mixed rat/human receptors)responses to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and0.05% monellin are shown. Rat T1R2/T1R3 does not respond to aspartame ormonellin, which are not palatable to rodents.

FIGS. 3 a-3 c present functional data for the human T1R2/T1R3 receptor.Intracellular calcium responses of HEK cells stably expressing Gal 5that are transiently transfected with human T1R1, T1R3, and T1R1/T1R3 tovarious concentrations of L-glutamate are shown in FIG. 3 a.Potentiation of the T1R1/T1R3 response by IMP is shown in FIG. 3 b.T1R1/T1R3 dose responses to L-glutamate and L-glutamate plus 0.2 mM IMPand correlated psychophysical detection thresholds (X-axis circles) areshown in FIG. 3 c.

FIGS. 4 a-4 b present immunofluorescence and FACS experiments thatdemonstrate that fusing the PDZIP peptide (SEQ ID No:1) to human T1R2enhanced its expression on the surface of HEK cells.

FIG. 5 presents automated fluorescence imaging data for cell lines thatstably express Gα15 and human T1R1/T1R3. L-glutamate dose responses weredetermined in the presence of 0.5 mM IMP.

FIG. 6 presents automated fluorescence imaging data for cell lines thatstably express Gal 5 and human T1R2/T1R3. Sucrose, D-tryptophan,saccharin, and aspartame dose responses are shown for on stable cellline.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus provides isolated nucleic acid molecules encodingtaste-cell-specific G protein-coupled receptors (“GPCR”), and thepolypeptides they encode. These nucleic acid molecules and thepolypeptides that they encode are members of the T1R family oftaste-cell-specific GPCRs. Members of the T1R family oftaste-cell-specific GPCRs are identified in Hoon et al., Cell,96:541-551 (1999), WO 00/06592, and WO 00/06593, all of which areincorporated herein by reference in their entireties.

More particularly, the invention provides nucleic acids encoding a novelfamily of taste-cell-specific GPCRs. These nucleic acids and thereceptors that they encode are referred to as members of the “T1R”family of taste-cell-specific GPCRs. In particular embodiments of theinvention, the T1R family members include human T1R1, T1R2, and T1R3. Asdescribed supra, different T1R combinations likely mediate sweet andumami taste.Further, it is believed that T1R family members may act in combinationwith other T1R family members, other taste-cell-specific GPCRs, or acombination thereof, to thereby effect chemosensory taste transduction.For instance, it is believed that T1R1 and T1R3 maybe coexressed withinthe same taste receptor cell type, and the two receptors may physicallyinteract to form a heterodimeric taste receptor. Alternatively, T1R1 andT1R3 may both independently bind to the same type of ligand, and theircombined binding may result in a specific perceived taste sensation.

These nucleic acids provide valuable probes for the identification oftaste cells, as the nucleic acids are specifically expressed in tastecells. For example, probes for T1R polypeptides and proteins can be usedto identify taste cells present in foliate, circumvallate, and fungiformpapillae, as well as taste cells present in the geschmackstreifen, oralcavity, gastrointestinal epithelium, and epiglottis. They may also serveas tools for the generation of taste topographic maps that elucidate therelationship between the taste cells of the tongue and taste sensoryneurons leading to taste centers in the brain. In particular, methods ofdetecting T1Rs can be used to identify taste cells sensitive to sweettaste stimuli or other specific modalities of taste stimuli.Furthermore, the nucleic acids and the proteins they encode can be usedas probes to dissect taste-induced behaviors. Also, chromosomelocalization of the genes encoding human T1Rs can be used to identifydiseases, mutations, and traits caused by and associated with T1R familymembers.

The nucleic acids encoding the T1R proteins and polypeptides of theinvention can be isolated from a variety of sources, geneticallyengineered, amplified, synthesized, and/or expressed recombinantlyaccording to the methods disclosed in WO 00/035,374, which is hereinincorporated by reference in its entirety.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel taste-cell-specific GPCRs. Such modulatorsof taste transduction are useful for pharmacological, chemical, andgenetic modulation of taste signaling pathways. These methods ofscreening can be used to identify high affinity agonists and antagonistsof taste cell activity. These modulatory compounds can then be used inthe food and pharmaceutical industries to customize taste, e.g., tomodulate the sweet tastes of foods or drugs.

Thus, the invention provides assays for detecting and characterizingtaste modulation, wherein T1R family members act as direct or indirectreporter molecules of the effect of modulators on taste transduction.GPCRs can be used in assays to, e.g., measure changes in ligand binding,ion concentration, membrane potential, current flow, ion flux,transcription, signal transduction, receptor-ligand interactions, secondmessenger concentrations, in vitro, in vivo, and ex vivo. In oneembodiment, members of the T1R family can be used as indirect reportersvia attachment to a second reporter molecule such as green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology, 15:961-964(1997)). In another embodiment, T1R family members may be recombinantlyexpressed in cells, and modulation of taste transduction via GPCRactivity may be assayed by measuring changes in Ca²⁺ levels and otherintracellular messages such as cAMP, cGMP, or IP3.

In certain embodiments, a domain of a T1R polypeptide, e.g., anextracellular, transmembrane, or intracellular domain, is fused to aheterologous polypeptide, thereby forming a chimeric polypeptide, e.g.,a chimeric polypeptide with GPCR activity. Such chimeric polypeptidesare useful, e.g., in assays to identify ligands, agonists, antagonists,or other modulators of a T1R polypeptide. In addition, such chimericpolypeptides are useful to create novel taste receptors with novelligand binding specificity, modes of regulation, signal transductionpathways, or other such properties, or to create novel taste receptorswith novel combinations of ligand binding specificity, modes ofregulation, signal transduction pathways, etc.

In one embodiment, a T1R polypeptide is expressed in a eukaryotic cellas a chimeric receptor with a heterologous, chaperone sequence thatfacilitates plasma membrane trafficking, or maturation and targetingthrough the secretory pathway. The optional heterologous sequence may bea rhodopsin sequence, such as an N-terminal fragment of a rhodopsin.Such chimeric T1R receptors can be expressed in any eukaryotic cell,such as HEK-293 cells. Preferably, the cells comprise a G protein, e.g.,Gα15 or Gα16 or another type of promiscuous G protein capable of pairinga wide range of chemosensory GPCRs to an intracellular signaling pathwayor to a signaling protein such as phospholipase C. Alternatively, thecells may express a chimeric or variant G protein that is selected basedon its ability to couple with T1Rs to produce a functional T1R tastereceptor. Examples of variant G proteins which are especially preferredinclude the G protein variants disclosed in U.S. Ser. No. 09/984,292,filed on Oct. 29, 2001, incorporated by reference herein in its entiretyand the chimeric Gal 5 variants disclosed in U.S. ProvisionalApplication No. ______, Attorney Docket No. 078003-0280737 alsoincorporated by reference in its entirety. These applications disclose Gprotein variants that have been shown to couple better with T1Rs thanGα15, a well known promiscuous G protein. Activation of such chimericreceptors in such cells can be detected using any standard method, suchas by detecting changes in intracellular calcium by detecting FURA-2dependent fluorescence in the cell. If preferred host cells do notexpress an appropriate G protein, they may be transfected with a geneencoding a promiscuous G protein such as those described in U.S.Application Serial No. 60/243,770, which is herein incorporated byreference in its entirety.

Methods of assaying for modulators of taste transduction include invitro ligand-binding assays using: T1R polypeptides, portions thereof,i.e., the extracellular domain, transmembrane region, or combinationsthereof, or chimeric proteins comprising one or more domains of a T1Rfamily member; oocyte or tissue culture cells expressing T1Rpolypeptides, fragments, or fusion proteins; phosphorylation anddephosphorylation of T1R family members; G protein binding to GPCRs;ligand-binding assays; voltage, membrane potential and conductancechanges; ion flux assays; changes in intracellular second messengerssuch as cGMP, CAMP and inositol triphosphate; changes in intracellularcalcium levels; and neurotransmitter release.

Further, the invention provides methods of detecting T1R nucleic acidand protein expression, allowing investigation of taste transductionregulation and specific identification of taste receptor cells. T1Rfamily members also provide useful nucleic acid probes for paternity andforensic investigations. T1R genes are also useful as a nucleic acidprobes for identifying taste receptor cells, such as foliate, fungiform,circumvallate, geschmackstreifen, and epiglottis taste receptor cells.T1R receptors can also be used to generate monoclonal and polyclonalantibodies useful for identifying taste receptor cells. Taste receptorcells can be identified using techniques such as reverse transcriptionand amplification of mRNA, isolation of total RNA or poly A+ RNA,northern blotting, dot blotting, in situ hybridization, RNaseprotection, S1 digestion, probing DNA microchip arrays, western blots,and the like.

Functionally, the T1R polypeptides comprise a family of related seventransmembrane G protein-coupled receptors, which are believed to beinvolved in taste transduction and may interact with a G protein tomediate taste signal transduction (see, e.g., Fong, Cell Signal, 8:217(1996); Baldwin, Curr. Opin. Cell Biol., 6:180 (1994)). Structurally,the nucleotide sequences of T1R family members may encode relatedpolypeptides comprising an extracellular domain, seven transmembranedomains, and a cytoplasmic domain. Related T1R family genes from otherspecies share at least about 50%, and optionally 60%, 70%, 80%, or 90%,nucleotide sequence identity over a region of at least about 50nucleotides in length, optionally 100, 200, 500, or more nucleotides inlength to SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, or conservativelymodified variants thereof, or encode polypeptides sharing at least about35 to 50%, and optionally 60%, 70%, 80%, or 90%, amino acid sequenceidentity over an amino acid region at least about 25 amino acids inlength, optionally 50 to 100 amino acids in length to SEQ ID NOS: 4, 10,12, 14, 17, 21, or conservatively modified variants thereof.

Several consensus amino acid sequences or domains have also beenidentified that are characteristic of T1R family members. For example,T1R family members typically comprise a sequence having at least about50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95-99%, orhigher, identity to T1R consensus sequences 1 and 2 (SEQ ID NOs 18 and19, respectively). These conserved domains thus can be used to identifymembers of the T1R family, by identity, specific hybridization oramplification, or specific binding by antibodies raised against adomain. Such T1R consensus sequences have the following amino acidsequences:

T1R Family Consensus Sequence 1: (SEQ ID NO: 18)(TR)C(FL)(RQP)R(RT)(SPV)(VERKT)FL(AE)(WL)(RHG)E T1R Family ConsensusSequence 2: (SEQ ID NO: 19)(LQ)P(EGT)(NRC)YN(RE)A(RK)(CGF)(VLI)T(FL)(AS)(ML)

These consensus sequences are inclusive of those found in the T1Rpolypeptides described herein, but T1R family members from otherorganisms may be expected to comprise consensus sequences having about75% identity or more to the inclusive consensus sequences describedspecifically herein.

Specific regions of the T1R nucleotide and amino acid sequences may beused to identify polymorphic variants, interspecies homologs, andalleles of T1R family members. This identification can be made in vitro,e.g., under stringent hybridization conditions or PCR (e.g., usingprimers encoding the T1R consensus sequences identified above), or byusing the sequence information in a computer system for comparison withother nucleotide sequences. Different alleles of T1R genes within asingle species population will also be useful in determining whetherdifferences in allelic sequences correlate to differences in tasteperception between members of the population. Classical PCR-typeamplification and cloning techniques are useful for isolating orthologs,for example, where degenerate primers are sufficient for detectingrelated genes across species, which typically have a higher level ofrelative identity than paralogous members of the T1R family within asingle species.

For instance, degenerate primers SAP077 (SEQ. ID NO. 5) and SAP0079(SEQ. ID NO. 6) can be used can be used to amplify and clone T1R3 genesfrom different mammalian genomes. In contrast, genes within a singlespecies that are related to T1R3 are best identified using sequencepattern recognition software to look for related sequences. Typically,identification of polymorphic variants and alleles of T1R family memberscan be made by comparing an amino acid sequence of about 25 amino acidsor more, e.g., 50-100 amino acids. Amino acid identity of approximatelyat least 35 to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99%,or above typically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of a T1R family member. Sequencecomparison can be performed using any of the sequence comparisonalgorithms discussed below. Antibodies that bind specifically to T1Rpolypeptides or a conserved region thereof can also be used to identifyalleles, interspecies homologs, and polymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of T1R genescan be confirmed by examining taste-cell-specific expression of theputative T1R polypeptide. Typically, T1R polypeptides having an aminoacid sequence disclosed herein can be used as a positive control incomparison to the putative T1R polypeptide to demonstrate theidentification of a polymorphic variant or allele of the T1R familymember. The polymorphic variants, alleles, and interspecies homologs areexpected to retain the seven transmembrane structure of a Gprotein-coupled receptor. For further detail, see WO 00/06592, whichdiscloses related T1R family members, GPCR-B3s, the contents of whichare herein incorporated by reference in a manner consistent with thisdisclosure. GPCR-B3 receptors are referred to herein as rT1R1 and mT1R1.Additionally, see WO 00/06593, which also discloses related T1R familymembers, GPCR-B4s, the contents of which are herein incorporated byreference in a manner consistent with this disclosure. GPCR-B4 receptorsare referred to herein as rT1R2 and mT1R2.

Nucleotide and amino acid sequence information for T1R family membersmay also be used to construct models of taste-cell-specific polypeptidesin a computer system. These models can be subsequently used to identifycompounds that can activate or inhibit T1R receptor proteins. Suchcompounds that modulate the activity of T1R family members can then beused to investigate the role of T1R genes and receptors in tastetransduction.

The present invention also provides assays, preferably high throughputassays, to identify molecules that interact with and/or modulate a T1Rpolypeptide. In numerous assays, a particular domain of a T1R familymember is used, e.g., an extracellular, transmembrane, or intracellulardomain or region. In numerous embodiments, an extracellular domain,transmembrane region or combination thereof may be bound to a solidsubstrate, and used, e.g., to isolate ligands, agonists, antagonists, orany other molecules that can bind to and/or modulate the activity of aT1R polypeptide.

In one aspect of the invention, a new human GPCR gene of the T1R family,termed hTIR3, is provided. The hT1R3 gene was identified from the humangenome sequence database including the HTGS division of GenBank. Thenucleotide and conceptually translated amino acid sequence for hT1R3 areprovided in SEQ. ID NOS 1-4. The hT1R3 receptor was identified in thepartially sequenced BAC genomic clone RP5-89003 (database accessionnumber AL139287) by virtue of its sequence similarity to the candidaterat taste receptor rT1R1 (accession number AF127389). By reference, thepairwise identity between the predicted hT1R3 and rT1R1 proteinsequences is approximately 34%. Sequence comparisons with additionalmembers of the GPCR Family C (which includes the calcium-sensingreceptors, putative V2R pheromone receptors, GABA-B receptors, fishtaste receptors, and metabotropic glutamate receptors) indicate thathT1R3 is likely to belong to the Family C subgroup defined by T1R1 and asecond rat candidate taste receptor (rT1R2, accession number AF127390).

The invention also provides the human ortholog, termed hT1R1, of a rattaste receptor, designated rT1R1. The gene products of rT1R1 and hT1R1are approximately 74% identical. The mouse gene, mT1R1 has beenreported, see Hoon et al., Cell, 96:541-551 (2000), and maps to achromosomal interval homologous to the interval containing hT1R1. Thenucleotide and conceptually-translated hT1R1 sequences are describedherein as SEQ. ID NOS 15 and 16, respectively.

While not wishing to be bound to any particular theory, the T1R familyof receptors is predicted to be involved in sweet taste transduction byvirtue of the linkage of mTIR3 to the Sac locus, a locus on the distalend of chromosome four that influences sweet taste. Human T1R3 has alsobeen reported to localize to 1p36.2-1p36.33, a region that displaysconserved synteny with the mouse interval containing Sac and T1R1.However, T1R type receptors may mediate other taste modalities, such asbitter, umami, sour and salty.

Various conservative mutations and substitutions are envisioned to bewithin the scope of the invention. For instance, it would be within thelevel of skill in the art to perform amino acid substitutions usingknown protocols of recombinant gene technology including PCR, genecloning, site-directed mutagenesis of cDNA, transfection of host cells,and in-vitro transcription. The variants could then be screened fortaste-cell-specific GPCR functional activity.

A. Identification and Characterization of T1R Polypeptides

The amino acid sequences of the T1R proteins and polypeptides of theinvention can be identified by putative translation of the codingnucleic acid sequences. These various amino acid sequences and thecoding nucleic acid sequences may be compared to one another or to othersequences according to a number of methods.

For example, in sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, as described below for the BLASTN andBLASTP programs, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403410(1990)). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare extended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and acomparison of both strands.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a so-called “tree” or “dendogram”showing the clustering relationships used to create the alignment (see,e.g., FIG. 2). PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395 (1984) encoded by the genes were derived by conceptualtranslation of the corresponding open reading frames. Comparison ofthese protein sequences to all known proteins in the public sequencedatabases using BLASTP algorithm revealed their strong homology to themembers of the T1R family, each of the T1R family sequences having atleast about 35 to 50%, and preferably at least 55%, at least 60%, atleast 65%, and most preferably at least 70%, amino acid identity to atleast one known member of the family.

B. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Taste cells” include neuroepithelial cells that are organized intogroups to form taste buds of the tongue, e.g., foliate, fungiform, andcircumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329-353 (1989)). Taste cells are also found in the palate and othertissues, such as the esophagus and the stomach.

“T1R” refers to one or more members of a family of G protein-coupledreceptors that are expressed in taste cells such as foliate, fungiform,and circumvallate cells, as well as cells of the palate, and esophagus(see, e.g., Hoon et al., Cell, 96:541-551 (1999), herein incorporated byreference in its entirety). Members of this family are also referred toas GPCR-B3 and TR1 in WO 00/06592 as well as GPCR-B4 and TR2 in WO00/06593. GPCR-B3 is also herein referred to as rT1R1, and GPCR-B4 isreferred to as rT1R2. Taste receptor cells can also be identified on thebasis of morphology (see, e.g., Roper, supra), or by the expression ofproteins specifically expressed in taste cells. T1R family members mayhave the ability to act as receptors for sweet taste transduction, or todistinguish between various other taste modalities.

“T1R” nucleic acids encode a family of GPCRs with seven transmembraneregions that have “G protein-coupled receptor activity,” e.g., they maybind to G proteins in response to extracellular stimuli and promoteproduction of second messengers such as IP3, cAMP, cGMP, and Ca²⁺ viastimulation of enzymes such as phospholipase C and adenylate cyclase(for a description of the structure and function of GPCRs, see, e.g.,Fong, supra, and Baldwin, supra). A single taste cell may contain manydistinct T1R polypeptides.

The term “T1R” family therefore refers to polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have at least about 35 to50% amino acid sequence identity, optionally about 60, 75, 80, 85, 90,95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NOS: 4,10, 12, 14, 17, or 21 over a window of about 25 amino acids, optionally50-100 amino acids; (2) specifically bind to antibodies raised againstan immunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 4, 10, 12, 14, 17, 21 and conservativelymodified variants thereof; (3) are encoded by a nucleic acid moleculewhich specifically hybridize (with a size of at least about 100,optionally at least about 500-1000 nucleotides) under stringenthybridization conditions to a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, andconservatively modified variants thereof; (4) comprise a sequence atleast about 35 to 50% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 4, 10, 12, 14, 17, or 21; or (5) areamplified by primers that specifically hybridize under stringenthybridization conditions to the same sequence as degenerate primer setsencoding SEQ ID NOS: 7, 8, and conservatively modified variants thereof.

Topologically, certain chemosensory GPCRs have an “N-terminal domain;”“extracellular domains;” “transmembrane domains” comprising seventransmembrane regions, and corresponding cytoplasmic, and extracellularloops; “cytoplasmic domains,” and a “C-terminal domain” (see, e.g., Hoonet al., Cell, 96:541-551 (1999); Buck & Axel, Cell, 65:175-187 (1991)).These domains can be structurally identified using methods known tothose of skill in the art, such as sequence analysis programs thatidentify hydrophobic and hydrophilic domains (see, e.g., Stryer,Biochemistry, (3rd ed. 1988); see also any of a number of Internet basedsequence analysis programs, such as those found atdot.imgen.bcm.tmc.edu). Such domains are useful for making chimericproteins and for in vitro assays of the invention, e.g., ligand-bindingassays.

“Extracellular domains” therefore refers to the domains of T1Rpolypeptides that protrude from the cellular membrane and are exposed tothe extracellular face of the cell. Such domains generally include the“N terminal domain” that is exposed to the extracellular face of thecell, and optionally can include portions of the extracellular loops ofthe transmembrane domain that are exposed to the extracellular face ofthe cell, i.e., the loops between transmembrane regions 2 and 3, betweentransmembrane regions 4 and 5, and between transmembrane regions 6 and7.

The “N terminal domain” region starts at the N-terminus and extends to aregion close to the start of the transmembrane domain. Moreparticularly, in one embodiment of the invention, this domain starts atthe N-terminus and ends approximately at the conserved glutamic acid atamino acid position 563 plus or minus approximately 20 amino acid. Theseextracellular domains are useful for in vitro ligand-binding assays,both soluble and solid phase. In addition, transmembrane regions,described below, can also bind ligand either in combination with theextracellular domain, and are therefore also useful for in vitroligand-binding assays.

“Transmembrane domain,” which comprises the seven “transmembraneregions,” refers to the domain of T1R polypeptides that lies within theplasma membrane, and may also include the corresponding cytoplasmic(intracellular) and extracellular loops. In one embodiment, this regioncorresponds to the domain of T1R family members which startsapproximately at the conserved glutamic acid residue at amino acidposition 563 plus or minus 20 amino acids and ends approximately at theconserved tyrosine amino acid residue at position 812 plus or minusapproximately 10 amino acids. The seven transmembrane regions andextracellular and cytoplasmic loops can be identified using standardmethods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32(1982)), or in Stryer, supra.

“Cytoplasmic domains” refers to the domains of T1R polypeptides thatface the inside of the cell, e.g., the “C terminal domain” and theintracellular loops of the transmembrane domain, e.g., the intracellularloop between transmembrane regions 1 and 2, the intracellular loopbetween transmembrane regions 3 and 4, and the intracellular loopbetween transmembrane regions 5 and 6. “C terminal domain” refers to theregion that spans the end of the last transmembrane domain and theC-terminus of the protein, and which is normally located within thecytoplasm. In one embodiment, this region starts at the conservedtyrosine amino acid residue at position 812 plus or minus approximately10 amino acids and continues to the C-terminus of the polypeptide.

The term “ligand-binding region” or “ligand-binding domain” refers tosequences derived from a chemosensory receptor, particularly a tastereceptor, that substantially incorporates at least the extracellulardomain of the receptor. In one embodiment, the extracellular domain ofthe ligand-binding region may include the N-terminal domain and,optionally, portions of the transmembrane domain, such as theextracellular loops of the transmembrane domain. The ligand-bindingregion may be capable of binding a ligand, and more particularly, ataste stimulus.

The phrase “functional effects” in the context of assays for testingcompounds that modulate T1R family member mediated taste transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the receptor, e.g., functional, physicaland chemical effects. It includes ligand binding, changes in ion flux,membrane potential, current flow, transcription, G protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivoand also includes other physiologic effects such increases or decreasesof neurotransmitter or hormone release.

By “determining the functional effect” in the context of assays is meantassays for a compound that increases or decreases a parameter that isindirectly or directly under the influence of a T1R family member, e.g.,functional, physical and chemical effects. Such functional effects canbe measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, oocyte T1R gene expression; tissue culture cell T1Rexpression; transcriptional activation of T1R genes; ligand-bindingassays; voltage, membrane potential and conductance changes; ion fluxassays; changes in intracellular second messengers such as cAMP, cGMP,and inositol triphosphate (IP3); changes in intracellular calciumlevels; neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of T1R genes or proteinsare used interchangeably to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays fortaste transduction, e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activators are compounds that, e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize, or up regulate taste transduction, e.g.,agonists. Modulators include compounds that, e.g., alter the interactionof a receptor with: extracellular proteins that bind activators orinhibitor (e.g., ebnerin and other members of the hydrophobic carrierfamily); G proteins; kinases (e.g., homologs of rhodopsin kinase andbeta adrenergic receptor kinases that are involved in deactivation anddesensitization of a receptor); and arrestins, which also deactivate anddesensitize receptors. Modulators can include genetically modifiedversions of T1R family members, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for inhibitors andactivators include, e.g., expressing T1R family members in cells or cellmembranes, applying putative modulator compounds, in the presence orabsence of taste stimuli, e.g., sweet taste stimuli, and thendetermining the functional effects on taste transduction, as describedabove. Samples or assays comprising T1R family members that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of modulation. Control samples (untreated withmodulators) are assigned a relative T1R activity value of 100%.Inhibition of a T1R is achieved when the T1R activity value relative tothe control is about 80%, optionally 50% or 25-0%. Activation of a T1Ris achieved when the T1R activity value relative to the control is 110%,optionally 150%, optionally 200-500%, or 1000-3000% higher.

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the state of being free of other, dissimilar compoundswith which the compound of the invention is normally associated in itsnatural state, so that the “purified,” “substantially purified,” and“isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, andmost preferably at least 50% or 75% of the mass, by weight, of a givensample. In one preferred embodiment, these terms refer to the compoundof the invention comprising at least 95% of the mass, by weight, of agiven sample. As used herein, the terms “purified,” “substantiallypurified,” and “isolated” “isolated,” when referring to a nucleic acidor protein, of nucleic acids or proteins, also refers to a state ofpurification or concentration different than that which occurs naturallyin the mammalian, especially human, body. Any degree of purification orconcentration greater than that which occurs naturally in the mammalian,especially human, body, including (1) the purification from otherassociated structures or compounds or (2) the association withstructures or compounds to which it is not normally associated in themammalian, especially human, body, are within the meaning of “isolated.”The nucleic acid or protein or classes of nucleic acids or proteins,described herein, may be isolated, or otherwise associated withstructures or compounds to which they are not normally associated innature, according to a variety of methods and processes known to thoseof skill in the art.

As used herein, the term “isolated,” when referring to a nucleic acid orpolypeptide refers to a state of purification or concentration differentthan that which occurs naturally in the mammalian, especially human,body. Any degree of purification or concentration greater than thatwhich occurs naturally in the body, including (1) the purification fromother naturally-occurring associated structures or compounds, or (2) theassociation with structures or compounds to which it is not normallyassociated in the body are within the meaning of “isolated” as usedherein. The nucleic acids or polypeptides described herein may beisolated or otherwise associated with structures or compounds to whichthey are not normally associated in nature, according to a variety ofmethods and processed known to those of skill in the art.

As used herein, the terms “amplifying” and “amplification” refer to theuse of any suitable amplification methodology for generating ordetecting recombinant or naturally expressed nucleic acid, as describedin detail, below. For example, the invention provides methods andreagents (e.g., specific degenerate oligonucleotide primer pairs) foramplifying (e.g., by polymerase chain reaction, PCR) naturally expressed(e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of theinvention (e.g., taste stimulus-binding sequences of the invention) invivo or in vitro.

The term “7-transmembrane receptor” means a polypeptide belonging to asuperfamily of transmembrane proteins that have seven domains that spanthe plasma membrane seven times (thus, the seven domains are called“transmembrane” or “TM” domains TM I to TM VII). The families ofolfactory and certain taste receptors each belong to this super-family.7-transmembrane receptor polypeptides have similar and characteristicprimary, secondary and tertiary structures, as discussed in furtherdetail below.

The term “library” means a preparation that is a mixture of differentnucleic acid or polypeptide molecules, such as the library ofrecombinantly generated chemosensory, particularly taste receptorligand-binding domains generated by amplification of nucleic acid withdegenerate primer pairs, or an isolated collection of vectors thatincorporate the amplified ligand-binding domains, or a mixture of cellseach randomly transfected with at least one vector encoding an tastereceptor.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones (see e.g., Oligonucleotides and Analogues, a PracticalApproach, ed. F. Eckstein, Oxford Univ. Press (1991); AntisenseStrategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds.Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993);Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997);Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, AntisenseNucleic Acid Drug Dev, 6:153-156 (1996)).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating, e.g., sequences in whichthe third position of one or more selected codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “plasma membrane translocation domain” or simply “translocationdomain” means a polypeptide domain that, when incorporated into theamino terminus of a polypeptide coding sequence, can with greatefficiency “chaperone” or “translocate” the hybrid (“fusion”) protein tothe cell plasma membrane. For instance, a “translocation domain” may bederived from the amino terminus of the bovine rhodopsin receptorpolypeptide, a 7-transmembrane receptor. However, rhodopsin from anymammal may be used, as can other translocation facilitating sequences.Thus, the translocation domain is particularly efficient intranslocating 7-transmembrane fusion proteins to the plasma membrane,and a protein (e.g., a taste receptor polypeptide) comprising an aminoterminal translocating domain will be transported to the plasma membranemore efficiently than without the domain. However, if the N-terminaldomain of the polypeptide is active in binding, the use of othertranslocation domains may be preferred.

The “translocation domain,” “ligand-binding domain”, and chimericreceptors compositions described herein also include “analogs,” or“conservative variants” and “mimetics” (“peptidomimetics”) withstructures and activity that substantially correspond to the exemplarysequences. Thus, the terms “conservative variant” or “analog” or“mimetic” refer to a polypeptide which has a modified amino acidsequence, such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity, asdefined herein. These include conservatively modified variations of anamino acid sequence, i.e., amino acid substitutions, additions ordeletions of those residues that are not critical for protein activity,or substitution of amino acids with residues having similar properties(e.g., acidic, basic, positively or negatively charged, polar ornon-polar, etc.) such that the substitutions of even critical aminoacids does not substantially alter structure and/or activity.

More particularly, “conservatively modified variants” applies to bothamino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein.

For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide.

Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin;ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr orile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (1); 5) Isoleucine (1), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton, Proteins, W. H. Freeman andCompany (1984); Schultz and Schimer, Principles of Protein Structure,Springer-Vrlag (1979)). One of skill in the art will appreciate that theabove-identified substitutions are not the only possible conservativesubstitutions. For example, for some purposes, one may regard allcharged amino acids as conservative substitutions for each other whetherthey are positive or negative. In addition, individual substitutions,deletions or additions that alter, add or delete a single amino acid ora small percentage of amino acids in an encoded sequence can also beconsidered “conservatively modified variations.”

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound that has substantially the same structural and/or functionalcharacteristics of the polypeptides, e.g., translocation domains,ligand-binding domains, or chimeric receptors of the invention. Themimetic can be either entirely composed of synthetic, non-naturalanalogs of amino acids, or may be a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also incorporate any amount of natural amino acidconservative substitutions as long as such substitutions also do notsubstantially alter the mimetic's structure and/or activity.

As with polypeptides of the invention which are conservative variants,routine experimentation will determine whether a mimetic is within thescope of the invention, i.e., that its structure and/or function is notsubstantially altered. Polypeptide mimetic compositions can contain anycombination of non-natural structural components, which are typicallyfrom three structural groups: a) residue linkage groups other than thenatural amide bond (“peptide bond”) linkages; b) non-natural residues inplace of naturally occurring amino acid residues; or c) residues whichinduce secondary structural mimicry, i.e., to induce or stabilize asecondary structure, e.g., a beta turn, gamma turn, beta sheet, alphahelix conformation, and the like. A polypeptide can be characterized asa mimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄), thiazole, retroamide, thioamide, orester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, NY (1983)). A polypeptide can also becharacterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues;non-natural residues are well described in the scientific and patentliterature.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid sequences thatdirect transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase 11 type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant means”also encompass the ligation of nucleic acids having various codingregions or domains or promoter sequences from different sources into anexpression cassette or vector for expression of, e.g., inducible orconstitutive expression of a fusion protein comprising a translocationdomain of the invention and a nucleic acid sequence amplified using aprimer of the invention.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridisation with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, optionally 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, Sx SSC, and 1% SDS, incubating at 42° C., or,Sx SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C. Such hybridizations and wash steps can be carried out for,e.g., 1, 2, 5, 10, 15, 30, 60; or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially related if the polypeptides whichthey encode are substantially related. This occurs, for example, when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-T1R” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a T1R gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or,“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a T1R family member from specific species such as rat, mouse,or human can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with the T1R polypeptide or animmunogenic portion thereof and not with other proteins, except fororthologs or polymorphic variants and alleles of the T1R polypeptide.This selection may be achieved by subtracting out antibodies thatcross-react with T1R molecules from other species or other T1Rmolecules. Antibodies can also be selected that recognize only T1R GPCRfamily members but not GPCRs from other families. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual, (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

The term “expression vector” refers to any recombinant expression systemfor the purpose of expressing a nucleic acid sequence of the inventionin vitro or in vivo, constitutively or inducibly, in any cell, includingprokaryotic, yeast, fungal, plant, insect or mammalian cell. The termincludes linear or circular expression systems. The term includesexpression systems that remain episomal or integrate into the host cellgenome. The expression systems can have the ability to self-replicate ornot, i.e., drive only transient expression in a cell. The term includesrecombinant expression “cassettes which contain only the minimumelements needed for transcription of the recombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

A. Isolation and Expression of T1R Polypeptides

Isolation and expression of the T1Rs, or fragments or variants thereof,of the invention can be performed as described below. PCR primers can beused for the amplification of nucleic acids encoding taste receptorligand-binding regions, and libraries of these nucleic acids canoptionally be generated. Individual expression vectors or libraries ofexpression vectors can then be used to infect or transfect host cellsfor the functional expression of these nucleic acids or libraries. Thesegenes and vectors can be made and expressed in vitro or in vivo. One ofskill will recognize that desired phenotypes for altering andcontrolling nucleic acid expression can be obtained by modulating theexpression or activity of the genes and nucleic acids (e.g., promoters,enhancers and the like) within the vectors of the invention. Any of theknown methods described for increasing or decreasing expression oractivity can be used. The invention can be practiced in conjunction withany method or protocol known in the art, which are well described in thescientific and patent literature.

The nucleic acid sequences of the invention and other nucleic acids usedto practice this invention, whether RNA, cDNA, genomic DNA, vectors,viruses or hybrids thereof, may be isolated from a variety of sources,genetically engineered, amplified, and/or expressed recombinantly. Anyrecombinant expression system can be used, including, in addition tomammalian cells, e.g., bacterial, yeast, insect, or plant systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411418 (1982);Adams, Am. Chem. Soc. 105:661 (1983); Belousov, Nucleic Acids Res.25:3440-3444 (1997); Frenkel, Free Radic. Biol. Med. 19:373-380 (1995);Blommers, Biochemistry 33:7886-7896 (1994); Narang, Meth. Enzymol. 68:90(1979); Brown, Meth. Enzymol. 68:109 (1979); Beaucage, Tetra. Lett.22:1859 (1981); U.S. Pat. No. 4,458,066. Double-stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Techniques for the manipulation of nucleic acids, such as, for example,for generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization and the like are well described in thescientific and patent literature. See, e.g., Sambrook, ed., MolecularCloning: a Laboratory manual (2nd ed.), Vols. 1-3, Cold Spring HarborLaboratory (1989); Current Protocols in Molecular Biology, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I, Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993).

Nucleic acids, vectors, capsids, polypeptides, and the like can beanalyzed and quantified by any of a number of general means well knownto those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatography, various immunological methods, e.g.,fluid or gel precipitin reactions, immunodiffusion,immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immuno-fluorescent assays, Southernanalysis, Northern analysis, dot-blot analysis, gel electrophoresis(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or targetor signal amplification methods, radiolabeling, scintillation counting,and affinity chromatography.

Oligonucleotide primers may be used to amplify nucleic acid fragmentsencoding taste receptor ligand-binding regions. The nucleic acidsdescribed herein can also be cloned or measured quantitatively usingamplification techniques. Amplification methods are also well known inthe art, and include, e.g., polymerase chain reaction, PCR(PCRProtocols, a Guide to Methods and Applications, ed. Innis. AcademicPress, N.Y. (1990) and PCR Strategies, ed. Innis, Academic Press, Inc.,N.Y. (1995), ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560(1989); Landegren, Science 241:1077 (1988); Barringer, Gene 89:117(1990)); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad.Sci. USA 86:1173 (1989)); and, self-sustained sequence replication (see,e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87:1874 (1990)); Q Betareplicase amplification (see, e.g., Smith, J. Clin. Microbiol.35:1477-1491 (1997)); automated Q-beta replicase amplification assay(see, e.g., Burg, Mol. Cell. Probes 10:257-271 (1996)) and other RNApolymerase mediated techniques (e.g., NASBA, Cangene, Mississauga,Ontario); see also Berger, Methods Enzymol. 152:307-316 (1987);Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan,Biotechnology 13:563-564 (1995). The primers can be designed to retainthe original sequence of the “donor” 7-membrane receptor. Alternatively,the primers can encode amino acid residues that are conservativesubstitutions (e.g., hydrophobic for hydrophobic residue, see abovediscussion) or functionally benign substitutions (e.g., do not preventplasma membrane insertion, cause cleavage by peptidase, cause abnormalfolding of receptor, and the like). Once amplified, the nucleic acids,either individually or as libraries, may be cloned according to methodsknown in the art, if desired, into any of a variety of vectors usingroutine molecular biological methods; methods for cloning in vitroamplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039.

The primer pairs may be designed to selectively amplify ligand-bindingregions of the T1R family members. These regions may vary for differentligands or taste stimuli. Thus, what may be a minimal binding region forone taste stimulus, may be too limiting for a second taste stimulus.Accordingly, ligand-binding regions of different sizes comprisingdifferent extracellular domain structures may be amplified.

Paradigms to design degenerate primer pairs are well known in the art.For example, a COnsensus-DEgenerate Hybrid Oligonucleotide Primer(CODEHOP) strategy computer program is accessible ashttp://blocks.fhcrc.org/codehop.html, and is directly linked from theBlockMaker multiple sequence alignment site for hybrid primer predictionbeginning with a set of related protein sequences, as known tastereceptor ligand-binding regions (see, e.g., Rose, Nucleic Acids Res.26:1628-1635 (1998); Singh, Biotechniques 24:318-319 (1998)).

Means to synthesize oligonucleotide primer pairs are well known in theart. “Natural” base pairs or synthetic base pairs can be used. Forexample, use of artificial nucleobases offers a versatile approach tomanipulate primer sequence and generate a more complex mixture ofamplification products. Various families of artificial nucleobases arecapable of assuming multiple hydrogen bonding orientations throughinternal bond rotations to provide a means for degenerate molecularrecognition. Incorporation of these analogs into a single position of aPCR primer allows for generation of a complex library of amplificationproducts. See, e.g., Hoops, Nucleic Acids Res. 25:4866-4871 (1997).Nonpolar molecules can also be used to mimic the shape of natural DNAbases. A non-hydrogen-bonding shape mimic for adenine can replicateefficiently and selectively against a nonpolar shape mimic for thymine(see, e.g., Morales, Nat. Struct. Biol. 5:950-954 (1998)). For example,two degenerate bases can be the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one or the purine baseN6-methoxy-2,6-diaminopurine (see, e.g., Hill, Proc. Natl. Acad. Sci.USA 95:4258-4263 (1998)). Exemplary degenerate primers of the inventionincorporate the nucleobase analog5′-Dimethoxytrityl-N-benzoyl-2′-deoxy-Cytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(the term “P” in the sequences, see above). This pyrimidine analoghydrogen bonds with purines, including A and G residues.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a taste receptor disclosed herein can beisolated using the nucleic acid probes described above. Alternatively,expression libraries can be used to clone T1R polypeptides andpolymorphic variants, alleles, and interspecies homologs thereof, bydetecting expressed homologs immunologically with antisera or purifiedantibodies made against a T1R polypeptide, which also recognize andselectively bind to the T1R homolog.

Nucleic acids that encode ligand-binding regions of taste receptors maybe generated by amplification (e.g., PCR) of appropriate nucleic acidsequences using degenerate primer pairs. The amplified nucleic acid canbe genomic DNA from any cell or tissue or mRNA or cDNA derived fromtaste receptor-expressing cells.

In one embodiment, hybrid protein-coding sequences comprising nucleicacids encoding T1Rs fused to a translocation sequences may beconstructed. Also provided are hybrid T1Rs comprising the translocationmotifs and taste stimulus-binding domains of other families ofchemosensory receptors, particularly taste receptors. These nucleic acidsequences can be operably linked to transcriptional or translationalcontrol elements, e.g., transcription and translation initiationsequences, promoters and enhancers, transcription and translationterminators, polyadenylation sequences, and other sequences useful fortranscribing DNA into RNA. In construction of recombinant expressioncassettes, vectors, and transgenics, a promoter fragment can be employedto direct expression of the desired nucleic acid in all desired cells ortissues.

In another embodiment, fusion proteins may include C-terminal orN-terminal translocation sequences. Further, fusion proteins cancomprise additional elements, e.g., for protein detection, purification,or other applications. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts,histidine-tryptophan modules, or other domains that allow purificationon immobilized metals; maltose binding protein; protein A domains thatallow purification on immobilized immunoglobulin; or the domain utilizedin the FLAGS extension/affinity purification system (Immunex Corp,Seattle Wash.).

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, Biochimie 80:289-293 (1998)), subtilisin proteaserecognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997));enterokinase (Invitrogen, San Diego, Calif.), and the like, between thetranslocation domain (for efficient plasma membrane expression) and therest of the newly translated polypeptide may be useful to facilitatepurification. For example, one construct can include a polypeptideencoding a nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin, an enterokinase cleavage site (see, e.g.,Williams, Biochemistry 34:1787-1797 (1995)), and an C-terminaltranslocation domain. The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the desired protein(s) from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see, e.g., Kroll, DNA Cell. Biol. 12:441-53 (1993).

Expression vectors, either as individual expression vectors or aslibraries of expression vectors, comprising the ligand-binding domainencoding sequences may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. See,e.g., Roberts, Nature 328:731 (1987); Berger supra; Schneider, ProteinExpr. Purif. 6435:10 (1995); Sambrook; Tijssen; Ausubel. Productinformation from manufacturers of biological reagents and experimentalequipment also provide information regarding known biological methods.The vectors can be isolated from natural sources, obtained from suchsources as ATCC or GenBank libraries, or prepared by synthetic orrecombinant methods.

The nucleic acids can be expressed in expression cassettes, vectors orviruses which are stably or transiently expressed in cells (e.g.,episomal expression systems). Selection markers can be incorporated intoexpression cassettes and vectors to confer a selectable phenotype ontransformed cells and sequences. For example, selection markers can codefor episomal maintenance and replication such that integration into thehost genome is not required. For example, the marker may encodeantibiotic resistance (e.g., chloramphenicol, kanamycin, G418,bleomycin, hygromycin) or herbicide resistance (e.g., chlorosulfuron orBasta) to permit selection of those cells transformed with the desiredDNA sequences (see, e.g., Blondelet-Rouault, Gene 190:315-317 (1997);Aubrecht, J. Pharmacol. Exp. Ther. 281:992-997 (1997)). Becauseselectable marker genes conferring resistance to substrates likeneomycin or hygromycin can only be utilized in tissue culture,chemoresistance genes are also used as selectable markers in vitro andin vivo.

A chimeric nucleic acid sequence may encode a T1R ligand-binding domainwithin any 7-transmembrane polypeptide. Because 7-transmembrane receptorpolypeptides have similar primary sequences and secondary and tertiarystructures, structural domains (e.g., extracellular domain, TM domains,cytoplasmic domain, etc.) can be readily identified by sequenceanalysis. For example, homology modeling, Fourier analysis and helicalperiodicity detection can identify and characterize the seven domainswith a 7-transmembrane receptor sequence. Fast Fourier Transform (FFT)algorithms can be used to assess the dominant periods that characterizeprofiles of the hydrophobicity and variability of analyzed sequences.Periodicity detection enhancement and alpha helical periodicity indexcan be done as by, e.g., Donnelly, Protein Sci. 2:55-70 (1993). Otheralignment and modeling algorithms are well known in the art, see, e.g.,Peitsch, Receptors Channels 4:161-164 (1996); Kyte & Doolittle, J. Md.Bio., 157:105-132 (1982); Cronet, Protein Eng. 6:59-64 (1993) (homologyand “discover modeling”); http://bioinfo.weizmann.ac.il/.

The present invention also includes not only the DNA and proteins havingthe specified nucleic and amino acid sequences, but also DNA fragments,particularly fragments of, e.g., 40, 60, 80, 100, 150, 200, or 250nucleotides, or more, as well as protein fragments of, e.g., 10, 20, 30,50, 70, 100, or 150 amino acids, or more. Optionally, the nucleic acidfragments can encode an antigenic polypeptide which is capable ofbinding to an antibody raised against a T1R family member. Further, aprotein fragment of the invention can optionally be an antigenicfragment which is capable of binding to an antibody raised against a T1Rfamily member.

Also contemplated are chimeric proteins, comprising at least 10, 20, 30,50, 70, 100, or 150 amino acids, or more, of one of at least one of theT1R polypeptides described herein, coupled to additional amino acidsrepresenting all or part of another GPCR, preferably a member of the 7transmembrane superfamily. These chimeras can be made from the instantreceptors and another GPCR, or they can be made by combining two or moreof the present receptors. In one embodiment, one portion of the chimeracorresponds tom or is derived from the extracellular domain of a T1Rpolypeptide of the invention. In another embodiment, one portion of thechimera corresponds to, or is derived from the extracellular domain andone or more of the transmembrane domains of a T1R polypeptide describedherein, and the remaining portion or portions can come from anotherGPCR. Chimeric receptors are well known in the art, and the techniquesfor creating them and the selection and boundaries of domains orfragments of G protein-coupled receptors for incorporation therein arealso well known. Thus, this knowledge of those skilled in the art canreadily be used to create such chimeric receptors. The use of suchchimeric receptors can provide, for example, a taste selectivitycharacteristic of one of the receptors specifically disclosed herein,coupled with the signal transduction characteristics of anotherreceptor, such as a well known receptor used in prior art assay systems.

For example, a domain such as a ligand-binding domain, an extracellulardomain, a transmembrane domain, a transmembrane domain, a cytoplasmicdomain, an N-terminal domain, a C-terminal domain, or any combinationthereof, can be covalently linked to a heterologous protein. Forinstance, an T1R extracellular domain can be linked to a heterologousGPCR transmembrane domain, or a heterologous GPCR extracellular domaincan be linked to a T1R transmembrane domain. Other heterologous proteinsof choice can include, e.g., green fluorescent protein, β-gal,glutamtate receptor, and the rhodopsin presequence.

Also within the scope of the invention are host cells for expressing theT1Rs, fragments, or variants of the invention. To obtain high levels ofexpression of a cloned gene or nucleic acid, such as cDNAs encoding theT1Rs, fragments, or variants of the invention, one of skill typicallysubclones the nucleic acid sequence of interest into an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. However, bacterial or eukaryotic expressionsystems can be used.

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al.) It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atlest one nucleic acid molecule into the host cell capable of expressingthe T1R, fragment, or variant of interest.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe receptor, fragment, or variant of interest, which is then recoveredfrom the culture using standard techniques. Examples of such techniquesare well known in the art. See, e.g., WO 00/06593, which is incorporatedby reference in a manner consistent with this disclosure.

B. Detection of T1R Polypeptides

In addition to the detection of T1R genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect T1Rs, e.g., to identify taste receptor cells, and variants of T1Rfamily members. Immunoassays can be used to qualitatively orquantitatively analyze the T1Rs. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

1. Antibodies to T1R family members

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with a T1R family member are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature, 256:495-497 (1975)). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science, 246:1275-1281 (1989);Ward et al., Nature, 341:544-546 (1989)).

A number of T1R-comprising immunogens may be used to produce antibodiesspecifically reactive with a T1R family member. For example, arecombinant T1R polypeptide, or an antigenic fragment thereof, can beisolated as described herein. Suitable antigenic regions include, e.g.,the consensus sequences that are used to identify members of the T1Rfamily. Recombinant proteins can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. For example, an inbred strain of mice (e.g., BALB/Cmice) or rabbits is immunized with the protein using a standardadjuvant, such as Freund's adjuvant, and a standard immunizationprotocol. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the T1R. When appropriately high titers of antibody to the immunogenare obtained, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired (see Harlow & Lane,supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen may be immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol., 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science, 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 104 or greater areselected and tested for their cross reactivity against non-T1Rpolypeptides, or even other T1R family members or other related proteinsfrom other organisms, using a competitive binding immunoassay. Specificpolyclonal antisera and monoclonal antibodies will usually bind with aKd of at least about 0.1 mM, more usually at least about 1 pM,optionally at least about 0.1 pM or better, and optionally 0.01 pM orbetter.

Once T1R family member specific antibodies are available, individual T1Rproteins and protein fragments can be detected by a variety ofimmunoassay methods. For a review of immunological and immunoassayprocedures, see Basic and Clinical Immunology (Stites & Terr eds., 7thed. 1991). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

2. Immunological Binding Assays

T1R proteins, fragments, and variants can be detected and/or quantifiedusing any of a number of well recognized immunological binding assays(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a review of the general immunoassays, see also Methodsin Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993);Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casea T1R family member or an antigenic subsequence thereof). The antibody(e.g., anti-T1R) may be produced by any of a number of means well knownto those of skill in the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled T1R polypeptide or alabeled anti-T1R antibody. Alternatively, the labeling agent may be athird moiety, such a secondary antibody, that specifically binds to theantibody/T1R complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol., 111:1401-1406 (1973); Akerstrom et al., J.Immunol., 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

a. Non-Competitive Assay Formats

Immunoassays for detecting a T1R polypeptide in a sample may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-T1R antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture the T1R polypeptide present in thetest sample. The T1R polypeptide is thus immobilized is then bound by alabeling agent, such as a second T1R antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second or thirdantibody is typically modified with a detectable moiety, such as biotin,to which another molecule specifically binds, e.g., streptavidin, toprovide a detectable moiety.

b. Competitive Assay Formats

In competitive assays, the amount of T1R polypeptide present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) T1R polypeptide displaced (competed away) from an anti-T1Rantibody by the unknown T1R polypeptide present in a sample. In onecompetitive assay, a known amount of T1R polypeptide is added to asample and the sample is then contacted with an antibody thatspecifically binds to the T1R. The amount of exogenous T1R polypeptidebound to the antibody is inversely proportional to the concentration ofT1R polypeptide present in the sample. In a particularly preferredembodiment, the antibody is immobilized on a solid substrate. The amountof T1R polypeptide bound to the antibody may be determined either bymeasuring the amount of T1R polypeptide present in a T1R/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed protein. The amount of T1R polypeptide may be detected byproviding a labeled T1R molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known T1R polypeptide is immobilized on a solidsubstrate. A known amount of anti-T1R antibody is added to the sample,and the sample is then contacted with the immobilized T1R. The amount ofanti-T1R antibody bound to the known immobilized T1R polypeptide isinversely proportional to the amount of T1R polypeptide present in thesample. Again, the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

c. Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcross-reactivity determinations. For example, a protein at leastpartially encoded by the nucleic acid sequences disclosed herein can beimmobilized to a solid support. Proteins (e.g., T1R polypeptides andhomologs) are added to the assay that compete for binding of theantisera to the immobilized antigen. The ability of the added proteinsto compete for binding of the antisera to the immobilized protein iscompared to the ability of the T1R polypeptide encoded by the nucleicacid sequences disclosed herein to compete with itself. The percentcross-reactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% cross-reactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs. In addition, peptides comprising amino acidsequences representing conserved motifs that are used to identifymembers of the T1R family can be used in cross-reactivitydeterminations.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a T1R familymember, to the immunogen protein (i.e., T1R polypeptide encoded by thenucleic acid sequences disclosed herein). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by nucleic acidsequences disclosed herein required to inhibit 50% of binding, then thesecond protein is said to specifically bind to the polyclonal antibodiesgenerated to a T1R immunogen.

Antibodies raised against T1R conserved motifs can also be used toprepare antibodies that specifically bind only to GPCRs of the T1Rfamily, but not to GPCRs from other families.

Polyclonal antibodies that specifically bind to a particular member ofthe T1R family can be made by subtracting out cross-reactive antibodiesusing other T1R family members. Species-specific polyclonal antibodiescan be made in a similar way. For example, antibodies specific to humanT1R1 can be made by, subtracting out antibodies that are cross-reactivewith orthologous sequences, e.g., rat T1R1 or mouse T1R1.

d. Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of T1R polypeptide in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the T1R polypeptide. The anti-T1R polypeptideantibodies specifically bind to the T1R polypeptide on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-T1Rantibodies.

Other, assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev., 5:34-41 (1986)).

e. Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

f. Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADSTM),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., 3H, 1251, 3sS, 14C, or³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize a T1Rpolypeptide, or secondary antibodies that recognize anti-T1R.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge-coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

C. Detection of Modulators

Compositions and methods for determining whether a test compoundspecifically binds to a chemosensory receptor of the invention, both invitro and in vivo, are described below. Many aspects of cell physiologycan be monitored to assess the effect of ligand binding to a T1Rpolypeptide of the invention. These assays may be performed on intactcells expressing a chemosensory receptor, on permeabilized cells, or onmembrane fractions produced by standard methods.

Taste receptors bind taste stimuli and initiate the transduction ofchemical stimuli into electrical signals. An activated or inhibited Gprotein will in turn alter the properties of target enzymes, channels,and other effector proteins. Some examples are the activation of cGMPphosphodiesterase by transducin in the visual system, adenylate cyclaseby the stimulatory G protein, phospholipase C by Gq and other cognate Gproteins, and modulation of diverse channels by Gi and other G proteins.Downstream consequences can also be examined such as generation ofdiacyl glycerol and IP3 by phospholipase C, and in turn, for calciummobilization by IP3.

The T1R proteins or polypeptides of the assay will typically be selectedfrom a polypeptide having a sequence of SEQ ID NOS: 4, 10, 12, 14, 17,21 or fragments or conservatively modified variants thereof. Optionally,the fragments and variants can be antigenic fragments and variants whichbind to an anti-T1R antibody.

Alternatively, the T1R proteins or polypeptides of the assay can bederived from a eukaryote host cell and can include an amino acidsubsequence having amino acid sequence identity to SEQ ID NOS: 4, 10,12, 14, 17, 21, or fragments or conservatively modified variantsthereof. Generally, the amino acid sequence identity will be at least 35to 50%, or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.Optionally, the T1R proteins or polypeptides of the assays can comprisea domain of a T1R protein, such as an extracellular domain,transmembrane region, transmembrane domain, cytoplasmic domain,ligand-binding domain, and the like. Further, as described above, theT1R protein or a domain thereof can be covalently linked to aheterologous protein to create a chimeric protein used in the assaysdescribed herein.

Modulators of T1R receptor activity are tested using T1R proteins orpolypeptides as described above, either recombinant or naturallyoccurring. The T1R proteins or polypeptides can be isolated, expressedin a cell, expressed in a membrane derived from a cell, expressed intissue or in an animal, either recombinant or naturally occurring. Forexample, tongue slices, dissociated cells from a tongue, transformedcells, or membranes can be used. Modulation can be tested using one ofthe in vitro or in vivo assays described herein.

1. In Vitro Binding Assays

Taste transduction can also be examined in vitro with soluble or solidstate reactions, using a T1R polypeptide of the invention. In aparticular embodiment, a T1R ligand-binding domain can be used in vitroin soluble or solid state reactions to assay for ligand binding.

For instance, the T1R N-terminal domain is predicted to be involved inligand binding. More particularly, the T1Rs belong to a GPCR sub-familythat is characterized by large, approximately 600 amino acid,extracellular N-terminal segments. These N-terminal segments are thoughtto form, at least in part, the ligand-binding domains, and are thereforeuseful in biochemical assays to identify T1R agonists and antagonists.The ligand-binding domain may also contain additional portions of theextracellular domain, such as the extracellular loops of thetransmembrane domain. Similar assays have been used with other GPCRsthat are related to the T1Rs, such as the metabotropic glutamatereceptors (see, e.g., Han and Hampson, J. Biol. Chem. 274:10008-10013(1999)). These assays might involve displacing a radioactively orfluorescently labeled ligand, measuring changes in intrinsicfluorescence or changes in proteolytic susceptibility, etc.

Ligand binding to a T1R polypeptide of the invention can be tested insolution, in a bilayer membrane, optionally attached to a solid phase,in a lipid monolayer, or in vesicles. Binding of a modulator can betested using, e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape),chromatographic, or solubility properties. Preferred binding assays ofthe invention are biochemical binding assays that use recombinantsoluble N-terminal T1R domains.

Receptor-G protein interactions can also be examined. For example,binding of the G protein to the receptor, or its release from thereceptor can be examined. More particularly, in the absence of GTP, anactivator will lead to the formation of a tight complex of a G protein(all three subunits) with the receptor. This complex can be detected ina variety of ways, as noted above. Such an assay can be modified tosearch for inhibitors, e.g., by adding an activator to the receptor andG protein in the absence of GTP, which form a tight complex, and thenscreen for inhibitors by looking at dissociation of the receptor-Gprotein complex. In the presence of GTP, release of the alpha subunit ofthe G protein from the other two G protein subunits serves as acriterion of activation. An activated or inhibited G protein will inturn alter the properties of target enzymes, channels, and othereffector proteins.

In another embodiment of the invention, a GTPγS assay may be used. Asdescribed above, upon activation of a GPCR, the Ga subunit of the Gprotein complex is stimulated to exchange bound GDP for GTP.Ligand-mediated stimulation of G protein exchange activity can bemeasured in a biochemical assay measuring the binding of addedradioactively-labeled GTPγ³⁵S to the G protein in the presence of aputative ligand. Typically, membranes containing the chemosensoryreceptor of interest are mixed with a complex of G proteins. Potentialinhibitors and/or activators and GTPγS are added to the assay, andbinding of GTPγS to the G protein is measured. Binding can be measuredby liquid scintillation counting or by any other means known in the art,including scintillation proximity assays (SPA). In other assays formats,fluorescently-labeled GTPγS can be utilized.

2. Fluorescence Polarization Assays

In another embodiment, Fluorescence Polarization (“FP”) based assays maybe used to detect and monitor ligand binding. Fluorescence polarizationis a versatile laboratory technique for measuring equilibrium binding,nucleic acid hybridization, and enzymatic activity. Fluorescencepolarization assays are homogeneous in that they do not require aseparation step such as centrifugation, filtration, chromatography,precipitation, or electrophoresis. These assays are done in real time,directly in solution and do not require an immobilized phase.Polarization values can be measured repeatedly and after the addition ofreagents since measuring the polarization is rapid and does not destroythe sample. Generally, this technique can be used to measurepolarization values of fluorophores from low picomolar to micromolarlevels. This section describes how fluorescence polarization can be usedin a simple and quantitative way to measure the binding of ligands tothe T1R polypeptides of the invention.

When a fluorescently labeled molecule is excited with plane polarizedlight, it emits light that has a degree of polarization that isinversely proportional to its molecular rotation. Large fluorescentlylabeled molecules remain relatively stationary during the excited state(4 nanoseconds in the case of fluorescein) and the polarization of thelight remains relatively constant between excitation and emission. Smallfluorescently labeled molecules rotate rapidly during the excited stateand the polarization changes significantly between excitation andemission. Therefore, small molecules have low polarization values andlarge molecules have high polarization values. For example, asingle-stranded fluorescein-labeled oligonucleotide has a relatively lowpolarization value but when it is hybridized to a complementary strand,it has a higher polarization value. When using FP to detect and monitortaste stimulus-binding which may activate or inhibit the chemosensoryreceptors of the invention, fluorescence-labeled taste stimuli orauto-fluorescent taste stimuli may be used.

Fluorescence polarization (P) is defined as:

$P = \frac{{Int}_{\coprod} - {Int}_{\bot}}{{Int}_{\coprod} + {Int}_{\bot}}$Where Π is the intensity of the emission light parallel to theexcitation light plane and Int ⊥ is the intensity of the emission lightperpendicular to the excitation light plane. P, being a ratio of lightintensities, is a dimensionless number. For example, the Beacon® andBeacon 2000™ System may be used in connection with these assays. Suchsystems typically express polarization in millipolarization units (1Polarization Unit=1000 mP Units).

The relationship between molecular rotation and size is described by thePerrin equation and the reader is referred to Jolley, M. E. (1991) inJournal of Analytical Toxicology, pp. 236-240, which gives a thoroughexplanation of this equation. Summarily, the Perrin equation states thatpolarization is directly proportional to the rotational relaxation time,the time that it takes a molecule to rotate through an angle ofapproximately 68.50 Rotational relaxation time is related to viscosity(η), absolute temperature (T), molecular volume (V), and the gasconstant (R) by the following equation:

${{Rotational}\mspace{14mu}{Relaxation}\mspace{14mu}{Time}} = \frac{3\;\eta\; V}{RT}$

The rotational relaxation time is small (≈1 nanosecond) for smallmolecules (e.g. fluorescein) and large (≈100 nanoseconds) for largemolecules (e.g. immunoglobulins). If viscosity and temperature are heldconstant, rotational relaxation time, and therefore polarization, isdirectly related to the molecular volume. Changes in molecular volumemay be due to interactions with other molecules, dissociation,polymerization, degradation, hybridization, or conformational changes ofthe fluorescently labeled molecule. For example, fluorescencepolarization has been used to measure enzymatic cleavage of largefluorescein labeled polymers by proteases, DNases, and RNases. It alsohas been used to measure equilibrium binding for protein/proteininteractions, antibody/antigen binding, and protein/DNA binding.

3. Solid State and Soluble High Throughput Assays

In yet another embodiment, the invention provides soluble assays using aT1R polypeptide; or a cell or tissue expressing an T1R polypeptide. Inanother embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the T1R polypeptide, or cellor tissue expressing the T1R polypeptide is attached to a solid phasesubstrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 1000 to about 1500different compounds. It is also possible to assay multiple compounds ineach plate well. It is possible to assay several different plates perday; assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention. More recently,microfluidic approaches to reagent manipulation have been developed.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non-covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders (see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc., 85:2149-2154 (1963) (describing solid phase synthesisof, e.g., peptides); Geysen et al., J. Immun. Meth., 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry, 39(4):718-719(1993); and Kozal et al., Nature Medicine, 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

4. Computer-Based Assays

Yet another assay for compounds that modulate T1R polypeptide activityinvolves computer assisted compound design, in which a computer systemis used to generate a three-dimensional structure of an T1R polypeptidebased on the structural information encoded by its amino acid sequence.The input amino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a T1R polypeptide intothe computer system. The nucleotide sequence encoding the T1Rpolypeptide, or the amino acid sequence thereof, can be any sequencedisclosed herein, and conservatively modified versions thereof.

The amino acid sequence represents the primary sequence or subsequenceof the protein, which encodes the structural information of the protein.At least 10 residues of the amino acid sequence (or a nucleotidesequence encoding 10 amino acids) are entered into the computer systemfrom computer keyboards, computer readable substrates that include, butare not limited to, electronic storage media (e.g., magnetic diskettes,tapes, cartridges, and chips), optical media (e.g., CD ROM), informationdistributed by internet sites, and by RAM. The three-dimensionalstructural model of the protein is then generated by the interaction ofthe amino acid sequence and the computer system, using software known tothose of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand-binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the T1R polypeptide to identify ligands that bind to theprotein. Binding affinity between the protein and ligands is determinedusing energy terms to determine which ligands have an enhancedprobability of binding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles, and interspecies homologs of T1R genes. Suchmutations can be associated with disease states or genetic traits. Asdescribed above, GeneChip™ and related technology can also be used toscreen for mutations, polymorphic variants, alleles, and interspecieshomologs. Once the variants are identified, diagnostic assays can beused to identify patients having such mutated genes. Identification ofthe mutated T1R genes involves receiving input of a first nucleic acidor amino acid sequence of a T1R gene, or conservatively modifiedversions thereof. The sequence is entered into the computer system asdescribed above. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in various T1R genes, and mutationsassociated with disease states and genetic traits.

5. Cell-Based Binding Assays

In one embodiment, a T1R protein or polypeptide is expressed in aeukaryotic cell as a chimeric receptor with a heterologous, chaperonesequence that facilitates its maturation and targeting through thesecretory pathway. Such chimeric T1R polypeptides can be expressed inany eukaryotic cell, such as HEK-293 cells. Preferably, the cellscomprise a functional G protein, e.g., Gα15, that is capable of couplingthe chimeric receptor to an intracellular signaling pathway or to asignaling protein such as phospholipase C. Activation of such chimericreceptors in such cells can be detected using any standard method, suchas by detecting changes in intracellular calcium by detecting FURA-2dependent fluorescence in the cell.

Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators will promote the transfer of 32P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature,10:349:117-27 (1991); Bourne et al., Nature, 348:125-32 (1990); Pitcheret al., Annu. Rev. Biochem., 67:653-92 (1998).

T1R modulation may be assayed by comparing the response of a T1Rpolypeptide treated with a putative T1R modulator to the response of anuntreated control sample. Such putative T1R modulators can include tastestimuli that either inhibit or activate T1R polypeptide activity. In oneembodiment, control samples (untreated with activators or inhibitors)are assigned a relative T1R activity value of 100. Inhibition of a T1Rpolypeptide is achieved when the T1R activity value relative to thecontrol is about 90%, optionally 50%, optionally 25-0%. Activation of aT1R polypeptide is achieved when the T1R activity value relative to thecontrol is 110%, optionally 150%, 200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes in ionicpolarization (i.e., electrical potential) of the cell or membraneexpressing a T1R polypeptide. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques(see, e.g., the “cell-attached” mode, the “inside-out” mode, and the“whole cell” mode, e.g., Ackerman et al., New Engl. J. Med.,336:1575-1595 (1997)). Whole cell currents are conveniently determinedusing the standard. Other known assays include: radiolabeled ion fluxassays and fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Gonzales& Tsien, Chem. Biol., 4:269-277 (1997); Daniel et al., J. Pharmacol.Meth., 25:185-193 (1991); Holevinsky et al., J. Membrane Biology,137:59-70 (1994)). Generally, the compounds to be tested are present inthe range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3, cGMP, orcAMP.

Preferred assays for GPCRs include cells that are loaded with ion orvoltage sensitive dyes to report receptor activity. Assays fordetermining activity of such receptors can also use known agonists andantagonists for other G protein-coupled receptors as negative orpositive controls to assess activity of tested compounds. In assays foridentifying modulatory compounds (e.g., agonists, antagonists), changesin the level of ions in the cytoplasm or membrane voltage will bemonitored using an ion sensitive or membrane voltage fluorescentindicator, respectively. Among the ion-sensitive indicators and voltageprobes that may be employed are those disclosed in the Molecular Probes1997 Catalog. For G protein-coupled receptors, promiscuous G proteinssuch as Gα15 and Gα16 can be used in the assay of choice (Wilkie et al.,Proc. Nat'l Acad. Sci., 88:10049-10053 (1991)). Such promiscuous Gproteins allow coupling of a wide range of receptors.

Receptor activation typically initiates subsequent intracellular events,e.g., increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some Gprotein-coupled receptors stimulates the formation of inositoltriphosphate (IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature, 312:315-21 (1984)). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G protein-coupledreceptor function. Cells expressing such G protein-coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Nat'l Acad. Sci.,88:9868-9872 (1991) and Dhallan et al., Nature, 347:184-187 (1990)). Incases where activation of the receptor results in a decrease in cyclicnucleotide levels, it may be preferable to expose the cells to agentsthat increase intracellular cyclic nucleotide levels, e.g., forskolin,prior to adding a receptor-activating compound to the cells in theassay. Cells for this type of assay can be made by co-transfection of ahost cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCRphosphatase and DNA encoding a receptor (e.g., certain glutamatereceptors, muscarinic acetylcholine receptors, dopamine receptors,serotonin receptors, and the like), which, when activated, causes achange in cyclic nucleotide levels in the cytoplasm.

In a preferred embodiment, T1R polypeptide activity is measured byexpressing a T1R gene in a heterologous cell with a promiscuous Gprotein that links the receptor to a phospholipase C signal transductionpathway (see Offermanns & Simon, J. Biol. Chem., 270:15175-15180(1995)). Optionally the cell line is HEK-293 (which does not naturallyexpress T1R genes) and the promiscuous G protein is Gα15 (Offermanns &Simon, supra). Modulation of taste transduction is assayed by measuringchanges in intracellular Ca²⁺ levels, which change in response tomodulation of the T1R signal transduction pathway via administration ofa molecule that associates with a T1R polypeptide. Changes in Ca²⁺levels are optionally measured using fluorescent Ca²⁺ indicator dyes andfluorometric imaging.

In one embodiment, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Bio. Chem., 270:15175-15180 (1995), may be used to determine thelevel of cAMP. Also, the method described in Felley-Bosco et al., Am. J.Resp. Cell and Mol. Biol., 11:159-164 (1994), may be used to determinethe level of cGMP. Further, an assay kit for measuring cAMP and/or cGMPis described in U.S. Pat. No. 4,115,538, herein incorporated byreference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with3H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist, to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist, to cpm in the presence of buffer control(which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining a T1R polypeptide of interest is contacted with a testcompound for a sufficient time to effect any interactions, and then thelevel of gene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by running a timecourse and measuring the level of transcription as a function of time.The amount of transcription may be measured by using any method known tothose of skill in the art to be suitable. For example, mRNA expressionof the protein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter gene may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, luciferase, ′3-galactosidase and alkalinephosphatase. Furthermore, the protein of interest can be used as anindirect reporter via attachment to a second reporter such as greenfluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology,15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the T1R polypeptide of interest.A substantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the T1R polypeptide of interest.

6. Transgenic Non-Human Animals Expressing Chemosensory Receptors

Non-human animals expressing one or more chemosensory receptor sequencesof the invention, can also be used for receptor assays. Such expressioncan be used to determine whether a test compound specifically binds to amammalian taste transmembrane receptor polypeptide in vivo by contactinga non-human animal stably or transiently transfected with a nucleic acidencoding a chemosensory receptor or ligand-binding region thereof with atest compound and determining whether the animal reacts to the testcompound by specifically binding to the receptor polypeptide.

Animals transfected or infected with the vectors of the invention areparticularly useful for assays to identify and characterize tastestimuli/ligands that can bind to a specific or sets of receptors. Suchvector-infected animals expressing human chemosensory receptor sequencescan be used for in vivo screening of taste stimuli and their effect on,e.g., cell physiology (e.g., on taste neurons), on the CNS, or behavior.

Means to infect/express the nucleic acids and vectors, eitherindividually or as libraries, are well known in the art. A variety ofindividual cell, organ, or whole animal parameters can be measured by avariety of means. The T1R sequences of the invention can be for exampleexpressed in animal taste tissues by delivery with an infecting agent,e.g., adenovirus expression vector.

The endogenous chemosensory receptor genes can remain functional andwild-type (native) activity can still be present. In other situations,where it is desirable that all chemosensory receptor activity is by theintroduced exogenous hybrid receptor, use of a knockout line ispreferred. Methods for the construction of non-human transgenic animals,particularly transgenic mice, and the selection and preparation ofrecombinant constructs for generating transformed cells are well knownin the art.

Construction of a “knockout” cell and animal is based on the premisethat the level of expression of a particular gene in a mammalian cellcan be decreased or completely abrogated by introducing into the genomea new DNA sequence that serves to interrupt some portion of the DNAsequence of the gene to be suppressed. Also, “gene trap insertion” canbe used to disrupt a host gene, and mouse embryonic stem (ES) cells canbe used to produce knockout transgenic animals (see, e.g., Holzschu,Transgenic Res 6:97-106 (1997)). The insertion of the exogenous istypically by homologous recombination between complementary nucleic acidsequences. The exogenous sequence is some portion of the target gene tobe modified, such as exonic, intronic or transcriptional regulatorysequences, or any genomic sequence which is able to affect the level ofthe target gene's expression; or a combination thereof. Gene targetingvia homologous recombination in pluripotential embryonic stem cellsallows one to modify precisely the genomic sequence of interest. Anytechnique can be used to create, screen for, propagate, a knockoutanimal, e.g., see Bijvoet, Hum. Mol. Genet. 7:53-62 (1998); Moreadith,J. Mol. Med. 75:208-216 (1997); Tojo, Cytotechnology 19:161-165 (1995);Mudgett, Methods Mol. Biol. 48:167-184 (1995); Longo, Transgenic Res.6:321-328 (1997); U.S. Pat. Nos. 5,616,491; 5,464,764; 5,631,153;5,487,992; 5,627,059; 5,272,071; WO 91/09955; WO93/09222; WO 96/29411;WO 95/31560; WO 91/12650.

The nucleic acids of the invention can also be used as reagents toproduce “knockout” human cells and their progeny. Likewise, the nucleicacids of the invention can also be used as reagents to produce“knock-ins” in mice. The human or rat T1R gene sequences can replace theorthologous T1R in the mouse genome. In this way, a mouse expressing ahuman or rat T1R is produced. This mouse can then be used to analyze thefunction of human or rat T1Rs, and to identify ligands for such T1Rs.

D. Modulators

The compounds tested as modulators of a T1R family member can be anysmall chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a T1R gene. Typically, test compoundswill be small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds can be dissolved in aqueousor organic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493(1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci., 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara etal., J. Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,114:9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),oligocarbamates (Cho et al., Science, 261:1303 (1993)), peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleicacid libraries (Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) andPCT/US96/10287), carbohydrate libraries (Liang et al., Science,274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993);thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pynrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS (Advanced Chem Tech, LouisvilleKy.), Symphony (Rainin, Woburn, Mass.), 433A (Applied Biosystems, FosterCity, Calif.), 9050 Plus (Millipore, Bedford, Mass.)). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences; Columbia, Md.; etc.).

In one aspect of the invention, the T1R modulators can be used in anyfood product, confectionery, pharmaceutical composition, or ingredientthereof to thereby modulate the taste of the product, composition, oringredient in a desired manner. For instance, T1R modulators whichenhance sweet taste sensation can be added to sweeten a product orcomposition, while T1R modulators which block undesirable tastesensations can be added to improve the taste of a product orcomposition.

E. Methods for Representing and Predicting the Perception of Taste

The invention also preferably provides methods for representing theperception of taste and/or for predicting the perception of taste in amammal, including in a human. Preferably, such methods may be performedby using the receptors and genes encoding said T1R polypeptidesdisclosed herein.

Also contemplated as within the invention, is a method of screening oneor more compounds for the presence of a taste detectable by a mammal,comprising: contacting said one or more compounds with the disclosedreceptors, preferably wherein the mammal is a human. Also contemplatedas within the invention, is a method for representing taste perceptionof a particular taste in a mammal, comprising the steps of: providingvalues Xi to Xn representative of the quantitative stimulation of eachof n chemosensory receptors of said vertebrate, where n is greater thanor equal to 2; and generating from said values a quantitativerepresentation of taste perception. The chemosensory receptors may be achemosensory receptor disclosed herein, the representation mayconstitutes a point or a volume in n-dimensional space, may constitutesa graph or a spectrum, and may constitutes a matrix of quantitativerepresentations. Also, the providing step may comprise contacting aplurality of recombinantly-produced chemosensory receptors with a testcomposition and quantitatively measuring the interaction of saidcomposition with said receptors.

Also contemplated as within the invention, is a method for predictingthe taste perception in a mammal generated by one or more molecules orcombinations of molecules yielding unknown taste perception in a mammal,comprising the steps of: providing values X₁ to X_(n) representative ofthe quantitative stimulation of each of n chemosensory receptors of saidvertebrate, where n is greater than or equal to 2, for one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal, providing values X₁ to X_(n) representative of thequantitative stimulation of each of n chemosensory receptors of saidvertebrate, where n is greater than or equal to 2, for one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal, and predicting the taste perception in a mammal generatedby one or more molecules or combinations of molecules yielding unknowntaste perception in a mammal by comparing the quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal to the quantitative representation of taste perception in amammal for the one or more molecules or combinations of moleculesyielding known taste perception in a mammal. The chemosensory receptorsused in this method may include a chemosensory receptor disclosedherein.

In another embodiment, novel molecules or combinations of molecules aregenerated which elicit a predetermined taste perception in a mammal bydetermining a value of taste perception in a mammal for a known moleculeor combinations of molecules as described above; determining a value oftaste perception in a mammal for one or more unknown molecules orcombinations of molecules as described above; comparing the value oftaste perception in a mammal for one or more unknown compositions to thevalue of taste perception in a mammal for one or more knowncompositions; selecting a molecule or combination of molecules thatelicits a predetermined taste perception in a mammal; and combining twoor more unknown molecules or combinations of molecules to form amolecule or combination of molecules that elicits a predetermined tasteperception in a mammal. The combining step yields a single molecule or acombination of molecules that elicits a predetermined taste perceptionin a mammal.

In another embodiment of the invention, there is provided a method forsimulating a taste, comprising the steps of: for each of a plurality ofcloned chemosensory receptors, preferably human receptors, ascertainingthe extent to which the receptor interacts with the taste stimulus; andcombining a plurality of compounds, each having a previously-ascertainedinteraction with one or more of the receptors, in amounts that togetherprovide a receptor-stimulation profile that mimics the profile for thetaste stimulus. Interaction of a taste stimulus with a chemosensoryreceptor can be determined using any of the binding or reporter assaysdescribed herein. The plurality of compounds may then be combined toform a mixture. If desired, one or more of the plurality of thecompounds can be combined covalently. The combined compoundssubstantially stimulate at least 75%, 80%, or 90% of the receptors thatare substantially stimulated by the taste stimulus.

In another preferred embodiment of the invention, a plurality ofstandard compounds are tested against a plurality of chemosensoryreceptors to ascertain the extent to which the receptors each interactwith each standard compound, thereby generating a receptor stimulationprofile for each standard compound. These receptor stimulation profilesmay then be stored in a relational database on a data storage medium.The method may further comprise providing a desired receptor-stimulationprofile for a taste; comparing the desired receptor stimulation profileto the relational database; and ascertaining one or more combinations ofstandard compounds that most closely match the desiredreceptor-stimulation profile. The method may further comprise combiningstandard compounds in one or more of the ascertained combinations tosimulate the taste.

F. Kits

T1R genes and their homologs are useful tools for identifyingchemosensory receptor cells, for forensics and paternity determinations,and for examining taste transduction. T1R family member-specificreagents that specifically hybridize to T1R nucleic acids, such as T1Rprobes and primers, and T1R specific reagents that specifically bind toa T1R polypeptide, e.g., T1R antibodies are used to examine taste cellexpression and taste transduction regulation.

Nucleic acid assays for the presence of DNA and RNA for a T1R familymember in a sample include numerous techniques are known to thoseskilled in the art, such as southern analysis, northern analysis, dotblots, RNase protection, S1 analysis, amplification techniques such asPCR, and in situ hybridization. In in situ hybridization, for example,the target nucleic acid is liberated from its cellular surroundings insuch as to be available for hybridization within the cell whilepreserving the cellular morphology for subsequent interpretation andanalysis. The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques, 4:230250 (1986); Haaseet al., Methods in Virology, vol. VII, pp.189-226 (1984); and NucleicAcid Hybridization: A Practical Approach (Names et al., eds. 1987). Inaddition, a T1R polypeptide can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing a recombinant T1Rpolypeptide) and a negative control.

The present invention also provides for kits for screening formodulators of T1R family members. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: T1R nucleic acids orproteins, reaction tubes, and instructions for testing T1R activity.Optionally, the kit contains a biologically active T1R receptor. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user.

EXAMPLES

In the protein sequences presented herein, the one-letter code X or Xaarefers to any of the twenty common amino acid residues. In the DNAsequences presented herein, the one letter codes N or n refers to any ofthe of the four common nucleotide bases, A, T, C, or G.

Example 1 hT1R3

The hT1R3 genomic DNA is provided below as SEQ ID NO 1 and SEQ ID NO 2with predicted coding sequences (cds) shown in boldface. The breakbetween the 5′ and 3′ contigs is shown as elipses (‘ . . . ’). The hT1R3predicted cds are described in SEQ ID NO 3. Finally, a preferred,predicted hT1R3 amino acid sequence is provided as SEQ ID NO 4, usingthe one-letter code for the amino acids.

hT1R3 genomic DNA-5′ contig (SEQ ID NO 1)AGCCTGGCAGTGGCCTCAGGCAGAGTCTGACGCGCA (SEQ ID NO: 1)CAAACTTTCAGGCCCAGGAAGCGAGGACACCACTGGGGCCCCAGGGTGTGGCAAGTGAGGATGGCAAGGGTTTTGCTAAACAAATCCTCTGCCCGCTCCCCGCCCCGGGCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTACAGAGGTGGGACGGCCTGGGTCGGGGTCAGGGTGACCAGGTCTGGGGTGCTCCTGAGCTGGGGCCGAGGTGGCCATCTGCGGTTCTGTGTGGCCCCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGTGGGGCGCCCCCCACCATCACCCACCCCCAACCAACCCCTGCCCCGTGGGAGCCCCTTGTG TCAGGAGAATGC hT1R3 genomicDNA—3′ contig (SEQ ID NO 2) ..................TACATGCACCCCACCCAG (SEQ IDNO 2) CCCTGCCCTGGGAGCCCTGTGTCAGAAGATGCTCTTGGCCTTGCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGGTGAGCCCGGGAGATGGGGGTGTGCTGTCCTCTGCATGTGCCCAGGCCACCAGGCACGGCCACCACGCCTGAGCTGGAGGTGGCTGGCGGCTCAGCCCCGTCCCCCGCCCGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGGTGAGGTGAGGGTGGGTGTGCCAGGCGTGCCCGTGGTAGCCCCCGCGGCAGGGCGCAGCCTGGGGGTGGGGGCCGTTCCAGTCTCCCGTGGGCATGCCCAGCCGAGCAGAGCCAGACCCCAGGCCTGTGCGCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGGTGAGCCGCCTTCCCGGCAGGCGGGGGTGGGAACGCAGCAGGGGAGGGTCCTGCCAAGTCCTGACTCTGAGACCAGAGCCCACAGGGTACAAGACGAACACCCAGCGCCCTTCTCCTCTCTCACAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATCTCAGCCCCGGTGAACCCAGACTTAGCTGCGATCCCCCCCAAGCCAGCAATGACCCGTGTCTCGCTACAGAGACCCTCCCGCTCTAGGTTCTGACCCCAGGTTGTCTCCTGACCCTGACCCCACAGTGAGCCCTAGGCCTGGAGCACGTGGACACCCCTGTGA CCATC hT1R3 full-length genomic DNA(SEQ ID NO 20) AGCCTGGCAGTGGCCTCAGGCAGAGTCTGACGCGCA (SEQ ID NO 20)CAAACTTTCAGGCCCAGGAAGCGAGGACACCACTGGGGCCCCAGGGTGTGGCAAGTGAGGATGGCAAGGGTTTTGCTAAACAAATCCTCTGCCCGCTCCCCGCCCCGGGCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTACAGAGGTGGGACGGCCTGGGTCGGGGTCAGGGTGACCAGGTCTGGGGTGCTCCTGAGCTGGGGCCGAGGTGGCCATCTGCGGTTCTGTGTGGCCCCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGTGGGGCGCCCCCCACCATCACCCACCCCCAACCAACCCCTGCCCCGTGGGAGCCCCTTGTGTCAGGAGAATGCTACATGCACCCCACCCAGCCCTGCCCTGGGAGCCCTGTGTCAGAAGATGCTCTTGGCCTTGCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGGTGAGCCCGGGAGATGGGGGTGTGCTGTCCTCTGCATGTGCCCAGGCCACCAGGCACGGCCACCACGCCTGAGCTGGAGGTGGCTGGCGGCTCAGCCCCGTCCCCCGCCCGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGGTGAGGTGAGGGTGGGTGTGCCAGGCGTGCCCGTGGTAGCCCCCGCGGCAGGGCGCAGCCTGGGGGTGGGGGCCGTTCCAGTCTCCCGTGGGCATGCCCAGCCGAGCAGAGCCAGACCCCAGGCCTGTGCGCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGGTGAGCCGCCTTCCCGGCAGGCGGGGGTGGGAACGCAGCAGGGGAGGGTCCTGCCAAGTCCTGACTCTGAGACCAGAGCCCACAGGGTACAAGACGAACACCCAGCGCCCTTCTCCTCTCTCACAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATCTCAGCCCCGGTGAACCCAGACTTAGCTGCGATCCCCCCCAAGCCAGCAATGACCCGTGTCTCGCTACAGAGACCCTCCCGCTCTAGGTTCTGACCCCAGGTTGTCTCCTGACCCTGACCCCACAGTGAGCCC TAGGCCTGGAGCACGTGGACACCCCTGTGACCATChT1R3 predicted cds (SEQ ID NO 3) ATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGG(SEQ ID NO 3) GCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAG TGA hT1R3 conceptual translation(SEQ ID NO 4) MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYV (SEQ ID NO 4)LGGLFPLGEAEEAGLRSRTRPSSPVCTRFSSNGLLWALAMKMAVEEINNKSDLLPGLRLGYDLFDTCSEPVVAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHSSELAMVTGKFFSFFLMPQVSYGASMELLSARETFPSFFRTVPSDRVQLTAAAELLQEFGWNWVAALGSDDEYGRQGLSIFSALAAARGICIAHEGLVPLPRADDSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWVASEAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALATDPAFCSALGEREQGLEEDWGQRCPQCDCITLQNVSAGLNHHQTFSVYAAVYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRFDSSGNVDMEYDLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDNQKPVSRCSRQCQEGQVRRVKGFHSCCYDCVDCEAGSYRQNPDDIACTFCGQDEWSPERSTRCFRRRSRFLAWGEPAVLLLLLLLSLALGLVLAALGLFVHHRDSPLVQASGGPLACFGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHLPLTGCLSTLFLQAAEIFVESELPLSWADRLSGCLRGPWAWLWLLAMLVEVALCTWYLVAFPPEVVTDWHMLPTEALVHCRTRSWVSFGLAHATNATLAFLCFLGTFLVRSQPGCYNRARGLTFAMLAYFITWVSFVPLLANVQWLRPAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLG GGPGDAQGQNDGNTGNQGKHE

Example 2 rT1R3 and mT1R3

Segments of the rat and mouse T1R3 genes were isolated by PCRamplification from genomic DNA using degenerate primers based on thehuman T1R3 sequence. The degenerate primers SAP077(5′-CGNTTYYTNGCNTGGGGNGARCC-3′; SEQ ID NO 5) and SAP079(5′-CGNGCNCGRTTRTARCANCCNGG-3′; SEQ ID NO 6) are complementary to humanT1R3 residues RFLAWGEPA (corresponding to SEQ ID NO 7) and PGCYNRAR(corresponding to SEQ ID NO 8), respectively. The PCR products werecloned and sequenced. Plasmid SAV115 carries a cloned segment of themouse T1R3 gene, and SAV118 carries a segment of the rat gene. Thesesequences, shown below, clearly represent the rodent counterparts ofhuman T1R3, since the mouse segment is 74% identical to thecorresponding segment of human T1R3, and the rat segment is 80%identical to the corresponding segment of human T1R3. The mouse and ratsegments are 88% identical. No other database sequences are more than40% identical to these T1R3 segments.

SAV115 mouse T1R3 segment in sense orientation (sequence correspondingto degenerate primer removed) (SEQ ID NO 9)GTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTG (SEQ ID NO 9)CTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGCGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGACTCTGGCCTGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGACCGCTTCACCAGAAGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAACCTGGGCTTGGTGCACATCACCMTGCAATGGTAGCTTTTCTCTGCTTTCTGGGCACTTTCCTGGTACAAGACCAG mT1R3 segment, conceptual translation(SEQ ID NO 10) VLSLLLLLCLVLGLALAALGLSVHHWDSPLVQASGGSQFCFGLICLGLFCLS (SEQID NO 10) VLLFPGRPSSASCLAQQPMAHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRDSGLLWLLATFVEAALCAWYLTASPEVVTDWSVLPTEVLEHCHVRSWVNLGLVHITNAMVAFLCFLGTFLVQDQ SAV118 rat T1R3 segment in sense orientation(sequence corresponding to degenerate primer removed) (SEQ ID NO 11)GTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGACACTGGCTG (SEQ ID NO 11)CCCTGGGGCTCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTTGCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGTGGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTACTGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGGCAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAGGGGTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAAAGCCAG rT1R3 segment, conceptualtranslation (SEQ ID NO 12)VLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASGGSLFCFGLICLGLFCLS (SEQ ID NO 12)VLLFPGRPRSASCLAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPWAWLWLLATLVEAALCAWYLMAFPPEWTDWQVLPTEVLEHCRMRSWVSLGLVHITNAGVAFLCFLGTFLVQSQ

Example 3 Cloning of rT1R3

The mT1R3 and rT1R3 fragments identified above as SEQ ID NOs 9 and 11were used to screen a rat taste tissue-derived cDNA library. Onepositive clone was sequenced and found to contain the full-length rT1R3sequence presented below as SEQ ID NO 13. Sequence comparison to themT1R3 and rT1R3 partial sequences and to the full-length hT1R3 sequenceestablished that this cDNA represents the rat counterpart to hT1R3. Forexample, the pairwise amino acid identity between rT1R3 and hT1R3 isapproximately 72%, whereas the most related annotated sequence in publicDNA sequence data banks is only approximately 33% identical to rT1R3.

rT1R3 predicted cds (SEQ. ID NO. 13)ATGCCGGGTTTGGCTATCTTGGGCCTCAGTCTGGCTGCTTTCCTGGAGCTTG (SEQ ID NO 13)GGATGGGGTCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTATATATTGGGTGGACTATTTCCCCTGGGCACAACTGAGGAGGCCACTCTCAACCAGAGAACACAGCCCAACGGCATCCTATGTACCAGGTTCTCGCCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTAGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGACTGGGCTATGACCTGTTTGACACATGCTCAGAGCCAGTGGTCACCATGAAGCCCAGCCTCATGTTCATGGCCAAGGTGGGAAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATTGGTCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGGGAAACATTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCCGTTGTGACACTGTTGCAGAATTTCAGCTGGAACTGGGTGGCTGCCTTAGGTAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTGGTCTGGCCAACTCACGAGGTATCTGCATTGCACACGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAATTGGGCAAGGTGGTGGATGTGCTACGCCAAGTGAACCAAAGCAAAGTACAGGTGGTGGTGCTGTTTGCATCTGCCCGTGCTGTCTACTCCCTTTTTAGCTACAGCATCCTTCATGACCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCCTGGCTGACCTCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTTCTTGGGTTTCTGCAGCGCGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCGCCTTGCCCTAGCTGCTGACCCAACATTCTGTGCCTCCCTGAAAGCTGAGTTGGATCTGGAGGAGCGCGTGATGGGGCCACGCTGTTCACAATGTGACTACATCATGCTACAGAACCTGTCATCTGGGCTGATGCAGAACCTATCAGCTGGGCAGTTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAGGCCCTTCACAACACCCTGCAGTGCAATGTCTCACATTGCCACACATCAGAGCCTGTTCAACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCGTGCTCGAGACTTGACACTGCAGTTTGATGCCAAAGGGAGTGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTACTACATACTGTAGGCACCTTCAACGGCACCCTTCAGCTGCAGCACTCGAAAATGTATTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGCTCCCGGCAGTGCAAAGATGGCCAGGTGCGCAGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGTGTGGACTGCAAGGCAGGGAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTGGCAAGGATCAGTGGTCCCCAGAAAAAAGCACAACCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGCTGTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGACACTGGCTGCCCTGGGGCTCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTTGCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGTGGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTACTGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGGCAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAGTGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGTCGCTATAACCGTGCCCGTGGCCTCACCTTCGCCATGCTAGCTTATTTCATCATCTGGGTCTCTTTTGTGCCCCTCCTGGCTAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCTTATTCTGTGCCCTGGGCATCCTGGCCACCTTCCACCTGCCCAAATGCTATGTACTTCTGTGGCTGCCAGAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAGCCCCAAGGAAGCATCAGATGGGAATAGTGGTAGTAGTGAGGCAACTCGGGGACACAGTGAATGA rT1R3 conceptual translation (SEQ. ID NO.14) MPGLAILGLSLAAFLELGMGSSLCLSQQFKAQGDYILGGLFPLGTTEEATLN (SEQ ID NO 14)QRTQPNGILCTRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKPSLMFMAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETEPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSGLANSRGICIAHEGLVPQHDTSGQQLGKVVDVLRQVNQSKVQVVVLFASARAVYSLFSYSILHDLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETRLALAADPTFCASLKAELDLEERVMGPRCSQCDYIMLQNLSSGLMQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHTSEPVQPWQLLENMYNMSFRARDLTLQFDAKGSVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQHSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCGKDQWSPEKSTTCLPRRPKFLAWGEPAVLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASGGSLFCFGLICLGLFCLSVLLFPGRPRSASCLAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPWAWLWLLATLVEAALCAWYLMAFPPEWTDWQVLPTEVLEHCRMRSWVSLGLVHITNAVLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFIIWVSFVPLLANVQVAYQPAVQMGAILFCALGILATFHLPKCYVLLWLPELNTQEFFLGRSPKEASDGNSGSSEATRGHSE

Example 4 Expression of mT1R3

The above described mouse T1R3 fragment contained in SAV115 was PCRamplified using M13 forward and M13 reverse primers and then gelpurified. The T1R3 DNA template was placed into an in vitrotranscription labeling reaction where Digoxigenin labeled UTP wasincorporated into an antisense cRNA probe. This probe was hybridized toadult mouse taste tissue containing cicumvallate papillae. The T1R3 insitu hybridization and detection were performed following the protocolof Schaeren-Wiemers et al., Histochemistry, 100:431-400 (1993). Briefly,fresh frozen mouse tongue was sectioned at 14 μm and prepared forhybridization. 200 ng/mL of the antisense Digoxigenin T1R3 probe washybridized for 14 hours at 72° C. Posthybridization consisted of a0.2×SSC wash at 72° C. Digoxigenin detection was accomplished byincubation with 1:5000 dilution of anti-DIG Alkaline Phosphataseantibody followed by a 12-hour reaction of the phosphatase in NBT/BCIP.

Example 5 hT1R1

The human ortholog (Database accession no. AL159177) of a rat tastereceptor, designated rT1R1, is provided below as SEQ ID NO 15. Predictedcds are indicated in bold and some intronic sequence intervals aredenoted as runs of N. The nucleotide and conceptually-translated hT1R1sequences are also described herein as SEQ ID NO 16 and 17, respectively

hT1R1 genomic DNA (SEQ ID NO 15)GAGAATCTCGCGAGATCCCGTCGGTCCGCCCCGCTGCCCTCCCAGCTGCCGA (SEQ ID NO 15)AAAGAGGGGCCTCCGAGCCGCCGGCGCCCTCTGCCGGCAACCTCCGGAAGCACACTAGGAGGTTCCAGCCGATCTGGTCGAGGGGCTCCACGGAGGACTCCATTTACGTTACGCAAATTCCCTACCCCAGCCGGCCGGAGAGAGAAAGCCAGAAACCTCGCGACCAGCCATGGGCCACCTCTCCGGAAAAACACCGGGATATTTTTTTTCTCCTGCAGAAAAAGCTTTAGGATTGGCAGTTTAAACAAAACATGTCTATTTGCATACCTTCGGTTTGCATGCATTTGTTTCGAAGTGAGCAACCCTGGGTAACAAGGCGAAAGTATATGACAATTTGCTCAGAATCTTAATGTCAGAAAACTGGAGACTGGGGCAGGGGGGTGTCGACTCAAAGCTGTGTCTCATTTAGTAAACTGAGGCCCAGGTAAAAAGTTCTGAAACCTCGCAACACCCGGAGAAATTGTGTTCCAGCCTCCCACCTCGCCCCAAAATGCCAGAGCTCCTTTTCTAAGCCAGGTGAAGTCACAGAGCGTGGACAGAACCCACAACCGTCCAGAGGAAGGGTCACTGGGTGCCACCTGGTTTGCATCTGTGCCTTCGTCCTGCCCAGTTCCTGAGTGGGACCGCAGGCCCGGAATGTCAAGGCAAACAGTCCTGCTTCAGCCACTGGGCTCCAGTCCCACCCCTTTTGGGGGCCTGAAGTTAGGAAGCATCCGGCAGCTGCCTTCTATTTAAGCAACTGGCCTCCTTAGAGGCCACTCCTTGGCCATGCCAGGCGCGGGCATCTGGCCAGCATGCTGCTCTGCACGGCTCGCCTGGTCGGCCTGCAGCTTCTCATTTCCTGCTGCTGGGCCTTTGCCTGCCATAGCACGGAGTCTTCTCCTGACTTCACCCTCCCCGGAGATTACCTCCTGGCAGGCCTGTTCCCTCTCCATTCTGGCTGTCTGCAGGTGAGGCACAGACCCGAGGTGACCCTGTGTGACAGGTGAGTGAGGGGCCAGCAGAGCCACACTTAGTGGGACCCCTGGCTATAGGGCCCCTCTGGCTGCCATCCTCCAAACAGGACCTTGCCTCTGCCTTTGCCCCTTGAACTGTCCCCAGGCCTTGTTCATCAATCCACTTGCCACCTAAGTGCTGGCTAGACCTTCCTAGACACTTCGGCCAGTTTCCAATTATTTCACCCTTGCTGTTAGAATGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAATTCCTTAAACTAAATTTCTCACTTTCTCTCTCTCTCTGGAAAACACTGACTAATGTAGCAGGTTTCTCTGCTCCAGGACTTCAGGACCTTTTCGATGCTAATAAGTTTCTCCATCAGGGCCAGCTTGTTCCTCCTACTGAGCTTGAGAGCCCTTGTTGAAGTTGTGGTTTGGGGGACTGGACCGATGACCTCAAAGGTTCCCTTTGCTCCCAAGCCTCAGAGTCTAGGAGGCCAGAGGGTCTCAGCAGGCCTTTGTCCTTCTCAGCTGTCTCTTACTGGCTTTCTCCACAGGTCTTGTAGCTTCAATGAGCATGGCTACCACCTCTTCCAGGCTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTACCAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCACATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTGCTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCCCATGGTAAGCTGGAGCCTCAGACCTTTGCCCATCTCCCTTCAGGCAAGTCTGGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCACCATGCCCGGCTAATTTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGCTGGTCGCAAACTCCTAACCTCGTGATCCACCCACCTCGGCCTCCCAATGTGCTGGGATTACAGGTGTGAGCCACTGCACCCGGCCATAATGTATTAATATAATAAAATAATTATACAACTCACCATAATGTAGAATCAGTGGGAGCCCTGAGCUGTTTTCCTACAACTAGATGGTCCCATCTGGGGGTGATGGGAGACAGTGACAGATCATCAGACATTAGATTCTCATAAGTAGCGTGCAACCCAGATCCCTCGCATGTGCAGTTCACAGTAGGGTTCAAGCTCCTACAAGAATCTGATGCTGCTGCTGATCTGACAGGAGGGGAGCAGCTGTAAATACAGATGAAGCTTCGCTTACTCACCAGCTGCTCACCTCCTCCTGTGAGGCCCGGTTCCTAACAGGCCACTGACCTAACTTCTGCCCTGACCTACACATGCTTCTCTTCTTCCTTGCAAACTGCCTCCAGTGGAAGTCCCTGAAGGTCCCCAAACACACGGGACTATTTCACTCCTATGCAGGTTTTGTCTCCTTTGCTTGGAATGCATCCCCTCACCCCTTGTCCCCAGGCAGATTCCCACCCCTCCCCCAGAACCTGCCCCAGTGGAGCCTTCGCAGGTGATTTGTCAGTTTCACAGGCTGAGGGGTGCTCTCCTGGTCTCCCCGGCTCCCTGTATCCCCACACCCAGCACAGGGCCAGGCACTGGGGGGGCCTTCAGTGGAGACTGAAATGGCTGAACGGGACCTCCCATAGATTAGCTATGCGGCCAGCAGCGAGACGCTCAGCGTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCTGCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGGAGAACCAGGCCACTGGTCAGGGGATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAGAGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGCCAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCTCCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCGTGGCCATCCAGAAGAGGGCTGTCCCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGAAGGCCCCTAGGCCTTGCCACAAGGGCTCCTGGTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCATGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCTGGAGCTTGTTCCAGGGGCCGAGTCTACCCCTGGCAGGTAAGAGAGCCCACCCCAGCACCTCCTGTCAGGGAGAACAGCCAATCCTGAGATGAGCAGAGTGGGCACTCTCCGGTCACTCTAAATGCCAAGGGGGATAAATGCCACTAACTTGAGGTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGACAGTCTGGCTCTGTCACCCAGGCTGCAGTGTAGTGATGCGATCTCGGCTCTCTGCAACTTCCACCTCCTGGGTTCAAGTGATTCTCTTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGCACCCACCACCATGCCTGGATAATTTTTCTTTTCTTTTTTTTTTTTTTGAGATAGAGTCTCGCTCTGTTGCCCAGGCTGGAATGCAGTGGTGCGATCTTGGCTCACTGTGAGCTCCGCCTCCCAGGTTCACTCCATTCCCCTGCCTCAGCCTCCCAAGTAGGTGGGACTACGGGCGCCCGCCACCACGCCCAGCTAATTTTTTTTGTATTTTGAGTAGAGACGGGGTTTCACCATGTTAGCCAGGATGGTCTCAATCTCCTGACCTTGTCATCCGCCCACCTCGTCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCACCCGGCCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGGCATCAAGTGATCCTCCTGCTTCGGCCTCCCAAAGTGCTGGGATTACAGGCATTAGCTCTCTTCTCTTAGACAGATCTTTCTCTCTGATCCTTGCCTTCTCTCACCCACTGTGTCTTGGAAGTGTCAAGTGATAAGATCCAGGGCTAAAACTGTCTGTAAAGGAGTGTTTGTTAGAGGCCTCCTCTCAGGAGGTTGGTGGGGAAGATTGAGGGGCTTCCTAAGAAGGAAGGGACGAGACCTTCCTGATGGGCTGAAACCACCAGGACGGAAACCCAGGAAGGCCCCAGGCCCTTGCTTCTGGGACCATGTGGGTCTGTGCTGTCTGTGGTGGCTTCATGATACGCGTTTCTTTCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAAGGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCAAGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGGCACGGAAAGGACAACCAGGTAATGGGGATGTGGCTACTCACCATGTAACTGGCTTATGGGCAACCTAGAGCCTGGGGGTGATGCTGACACAGTGTACAGGGAGCAGGAGGGGGGCCCCAGGGGTCCAGCTGCCACCACTCTACCCATCCTGGCCAGGGAAGCAGGGAAGACACTCCGTAGGCGAGTGTGCAGATGCCCTGGGGCGGAAGTTCACACGACCAGGGGCCCTGCCCTGGGAGTGAGCCCTGAGGGCAGATGCACAGAGATTCTGTTTTCTGTTCCACATGTGAGCTGTCCTTTGACTTGGGCCCCTACGTGTGGCCCCTCTGGCTTCTTACAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGGTTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGTGGTGAGTGGGCAATGGAGCAGGCGAGCTACCCAGCACTCCCGGGGGCTGCACGGTGGAGGGAGGGCCTCCCTTGGGCCCCATGTGCCCTGCCCCAGAACCAAGGCCCAGTCACTGGGCTGCCAGTTAGCTTCAGGTTGGAGGACACCTGCTACCAGACAGAATTCTGATCAAGAGAATCAGCCACTGGGTGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTGGATCACTTGAGGTCGGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACCAAAAATATAAAAAATTAGCTGGGTGTGGTGGCGCGTGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCCAAGATGCATTCCAGCCTGGACCACAAAGCGAGAATTCGTCCCCCCAAAAAAAGAAAGGAGGCCGGGCGCGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAAAAGTTAGCCGGGCGTTGTGGCGTGTGCCTGTAATTCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGCACCATTGCACTCCAGCCTGGGCGACAAGAGAAAAACTCTGTCTCAAAAAAAAAGAAAGAAAGAAAGAATTAGCCAACTGAAAGCCTTAGACTGAGGTGTGTCCTCTGTTAGAGAGCTGTCATCACAACTCCTACAAAAGCAGTCGTATCCTGAATTCAACCTCTTTCTCTAAATGAATATAGCTATTGTTCCCTTTGTGCCCTCTTGTCCTACTGTCCCCTTCTGTTGCCCATGCCAAAGACAGCTAGCTCCTTGAACAGCTTGGCCTGAATACAGATACTAGCGTGTCTGCAGCAGAGAAAAAAACAGCATTCCCCATCCAGAAATGCAAGGTCAAGAACAGAGAGCAAATTAGGTAGCTAAGGACTCAGGTCCTTAGTTGGTGTCCAGGGGCCACATTCTTTCCTTTCACCATCTCTGTAGGGACAGGAATACTTCCCTTCTGTCCTCAGAGGGTCAGGACTCAGAGAAACCACAGAGCAGCAGCTCAGGAAAGTGGTTCATGGAAATGCTGGCAAGAGAGAGGGGTTACAATGCCCTCCCTTGGGAGCAGGCTGCTCCCATCAGATCGTAACCTCTCTGGTATGTGGGCAGAGCTACCAGGTTAAGGTCCTCCCTAGGGTTTGCAAAACCCTCATGGGATCATGAGCCATACAGAACCGACCTGTGTGTCTCCAGAGTCTGTAATTAACACAGGCATTTTGAGGAAATGCGTGGCCTCAGGCCCCACTCCCGGCTACCCCCATCCCACTATGCCTAGTATAGTCTAGCTGCCCTGGTACAATTCTCCCAGTATCTTGCAGGCCCCTATTTCCTATTCCTACTCTGCTCATCTGGCTCTCAGGAACCTTCTTGGCCTTCCCTTTCAGACCTCTACAGATGCCAGCCTTGTGGGAAAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTGCGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTTTGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAGCAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCCCTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAAGGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATGATCAGCTCAGCGGCCCAGCTGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTGATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGCCTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCTTCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATGATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCCAGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGACCAGTGGGTCAGCAGGCACGGCTGGCAGCCTTCTCTGCCCTGAGGGTCGAAGGTCGAGCAGGCCGGGGGTGTCCGGGAGGTCTTTGGGCATCGCGGTCTGGGGTTGGGACGTGTAAGCGCCTGGGAGAGCCTAGACCAGGCTCCGGGCTGCCAATAAAGAAGTGAAATGCGTATCTGGTCTCCTGTCGTGGGAGAGTGTGAGGTGTAACGGATTCAAGTCTGAACCCAGAGCCTGGAAAAGGCTGACCGCCCAGATTGACGTTGCTAGGCAACTCCGGAGGCGGGCCCAGCGCCAAAAGAACAGGGCGAGGCGTCGTCCCCGCATCCCATTGGCCGTTCTCTGCGGGGCCCCGCCCTCGGGGGCCGGAGCTAGAAGCTCTACGCTTCCGAGGCGCACCTCCTGGCCTGCACGCTTTGACGT hT1R1 predicted cds (SEQ ID NO 16)ATGCTGCTCTGCACGGCTCGCCTGGTCGGCCTGCAGCTTCTCATTTCCTGCT (SEQ ID NO 16)GCTGGGCCTTTGCCTGCCATAGCACGGAGTCTTCTCCTGACTTCACCCTCCCCGGAGATTACCTCCTGGCAGGCCTGTTCCCTCTCCATTCTGGCTGTCTGCAGGTGAGGCACAGACCCGAGGTGACCCTGTGTGACAGGTCTTGTAGCTTCAATGAGCATGGCTACCACCTCTTCCAGGCTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTACCAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCACATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTGCTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCCCATGATTAGCTATGCGGCCAGCAGCGAGACGCTCAGCGTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCTGCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGGAGAACCAGGCCACTGGTCAGGGGATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAGAGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGCCAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCTCCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCGTGGCCATCCAGAAGAGGGCTGTCCCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGAAGGCCCCTAGGCCTTGCCACAAGGGCTCCTGGTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCATGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCTGGAGCTTGTTCCAGGGGCCGAGTCTACCCCTGGCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAAGGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCAAGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGGCACGGAAAGGACAACCAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGGTTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGTGACCTCTACAGATGCCAGCCTTGTGGGAAAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTGCGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTTTGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAGCAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCCCTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAAGGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATGATCAGCTCAGCGGCCCAGCTGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTGATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGCCTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCTTCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATGATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCCAGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGA hT1R1 conceptual translation (SEQ ID NO17) MLLCTARLVGLQLLISCCWAFACHSTESSPDFTLPGDYLLAGLFPLHSGCLQ (SEQ ID NO 17)VRHRPEVTLCDRSCSFNEHGYHLFQAMRLGVEEINNSTALLPNITLGYQLYDVCSDSANVYATLRVLSLPGQHHIELQGDLLHYSPTVLAVIGPDSTNRAATTAALLSPFLVPMISYAASSETLSVKRQYPSFLRTIPNDKYQVETMVLLLQKFGWTWISLVGSSDDYGQLGVQALENQATGQGICIAFKDIMPFSAQVGDERMQCLMRHLAQAGATVWVFSSRQLARVFFESWLTNLTGKVWVASEAWALSRHITGVPGIQRIGMVLGVAIQKRAVPGLKAFEEAYARADKKAPRPCHKGSWCSSNQLCRECQAFMAHTMPKLKAFSMSSAYNAYRAVYAVAHGLHQLLGCASGACSRGRVYPWQLLEQIHKVHFLLHKDTVAFNDNRDPLSSYNIIAWDWNGPKWTFTVLGSSTWSPVQLNINETKIQWHGKDNQVPKSVCSSDCLEGHQRWTGFHHCCFECVPCGAGTFLNKSDLYRCQPCGKEEWAPEGSQTCFPRTVVFLALREHTSWVLLAANTLLLLLLLGTAGLFAWHLDTPWRSAGGRLCFLMLGSLAAGSGSLYGFFGEPTRPACLLRQALFALGFTIFLSCLTVRSFQLIIIFKFSTKVPTFYHAWVQNHGAGLFVMISSAAQLLICLTWLVVWTPLPAREYQRFPHLVMLECTETNSLGFILAFLYNGLLSISAFACSYLGKDLPENYNEAKCVTFSLLFNFVSWIAFFTTASVYDGKYLPAANMMAGLSSLSSGFGGYFLPKCWILCRPDLNSTEHFQASIQDYTRR CGST

Example 6 hT1R2

The predicted cds of the human ortholog of a rat taste receptor,designated rT1R2, is provided below as SEQ ID NO 20. Theconceptually-translated hT1R2 sequences is also described herein as SEQID NO 21. According to the present invention, the first two coding exonsof hT1R2 were identified within a PAC by Southern blot. Exon 1 wasisolated within a BamHI/BglII fragment that was identified in theSouthern experiment, and exon 2 was isolated within a PCR product thatspans exon 1 to exon 3. Comparison of the first two coding exons to therT1R2 sequence established that the two exons encode the N-terminus ofthe human counterpart to rT1R2. For example, the pairwise amino acididentity between the hT1R2 N-terminal sequence coded by the two exonsand corresponding regions of rT1R2 is approximately 72%, whereas themost related annotated sequence in public DNA sequence data banks isonly approximately 48% identical to hT1R2.

hT1R2 predicted cds (SEQ ID NO 20)ATGGGGCCCAGGGCAAAGACCATCTGCTCCCTGTTCTTCCTCCTATGGGTCC (SEQ ID NO. 20)TGGCTGAGCCGGCTGAGAACTCGGACTTCTACCTGCCTGGGGATTACCTCCTGGGTGGCCTCTTCTCCCTCCATGCCAACATGAAGGGCATTGTTCACCTTAACTTCCTGCAGGTGCCCATGTGCAAGGAGTATGAAGTGAAGGTGATAGGCTACAACCTCATGCAGGCCATGCGCTTCGCGGTGGAGGAGATCAACAATGACAGCAGCCTGCTGCCTGGTGTGCTGCTGGGCTATGAGATCGTGGATGTGTGCTACATCTCCAACAATGTCCAGCCGGTGCTCTACTTCCTGGCACACGAGGACAACCTCCTTCCCATCCAAGAGGACTACAGTAACTACATTTCCCGTGTGGTGGCTGTCATTGGCCCTGACAACTCCGAGTCTGTCATGACTGTGGCCAACTTCCTCTCCCTATTTCTCCTTCCACAGATCACCTACAGCGCCATCAGCGATGAGCTGCGAGACAAGGTGCGCTTCCCGGCTTTGCTGCGTACCACACCCAGCGCCGACCACCACGTCGAGGCCATGGTGCAGCTGATGCTGCACTTCCGCTGGAACTGGATCATTGTGCTGGTGAGCAGCGACACCTATGGCCGCGACAATGGCCAGCTGCTTGGCGAGCGCGTGGCCCGGCGCGACATCTGCATCGCCTTCCAGGAGACGCTGCCCACACTGCAGCCCAACCAGAACATGACGTCAGAGGAGCGCCAGCGCCTGGTGACCATTGTGGACAAGCTGCAGCAGAGCACAGCGCGCGTCGTGGTCGTGTTCTCGCCCGACCTGACCCTGTACCACTTCTTCAATGAGGTGCTGCGCCAGAACTTCACGGGCGCCGTGTGGATCGCCTCCGAGTCCTGGGCCATCGACCCGGTCCTGCACAACCTCACGGAGCTGGGCCACTTGGGCACCTTCCTGGGCATCACCATCCAGAGCGTGCCCATCCCGGGCTTCAGTGAGTTCCGCGAGTGGGGCCCACAGGCTGGGCCGCCACCCCTCAGCAGGACCAGCCAGAGCTATACCTGCAACCAGGAGTGCGACAACTGCCTGAACGCCACCTTGTCCTTCAACACCATTCTCAGGCTCTCTGGGGAGCGTGTCGTCTACAGCGTGTACTCTGCGGTCTATGCTGTGGCCCATGCCCTGCACAGCCTCCTCGGCTGTGACAAAAGCACCTGCACCAAGAGGGTGGTCTACCCCTGGCAGCTGCTTGAGGAGATCTGGAAGGTCAACTTCACTCTCCTGGACCACCAAATCTTCTTCGACCCGCAAGGGGACGTGGCTCTGCACTTGGAGATTGTCCAGTGGCAATGGGACCGGAGCCAGAATCCCTTCCAGAGCGTCGCCTCCTACTACCCCCTGCAGCGACAGCTGAAGAACATCCAAGACATCTCCTGGCACACCGTCAACAACACGATCCCTATGTCCATGTGTTCCAAGAGGTGCCAGTCAGGGCAAAAGAAGAAGCCTGTGGGCATCCACGTCTGCTGCTTCGAGTGCATCGACTGCCTTCCCGGCACCTTCCTCAACCACACTGAAGATGAATATGAATGCCAGGCCTGCCCGAATAACGAGTGGTCCTACCAGAGTGAGACCTCCTGCTTCAAGCGGCAGCTGGTCTTCCTGGAATGGCATGAGGCACCCACCATCGCTGTGGCCCTGCTGGCCGCCCTGGGCTTCCTCAGCACCCTGGCCATCCTGGTGATATTCTGGAGGCACTTCCAGACACCCATAGTTCGCTCGGCTGGGGGCCCCATGTGCTTCCTGATGCTGACACTGCTGCTGGTGGCATACATGGTGGTCCCGGTGTACGTGGGGCCGCCCAAGGTCTCCACCTGCCTCTGCCGCCAGGCCCTCTTTCCCCTCTGCTTCACAATTTGCATCTCCTGTATCGCCGTGCGTTCTTTCCAGATCGTCTGCGCCTTCAAGATGGCCAGCCGCTTCCCACGCGCCTACAGCTACTGGGTCCGCTACCAGGGGCCCTACGTCTCTATGGCATTTATCACGGTACTCAAAATGGTCATTGTGGTAATTGGCATGCTGGCCACGGGCCTCAGTCCCACCACCCGTACTGACCCCGATGACCCCAAGATCACAATTGTCTCCTGTAACCCCAACTACCGCAACAGCCTGCTGTTCAACACCAGCCTGGACCTGCTGCTCTCAGTGGTGGGTTTCAGCTTCGCCTACATGGGCAAAGAGCTGCCCACCAACTACAACGAGGCCAAGTTCATCACCCTCAGCATGACCTTCTATTTCACCTCATCCGTCTCCCTCTGCACCTTCATGTCTGCCTACAGCGGGGTGCTGGTCACCATCGTGGACCTCTTGGTCACTGTGCTCAACCTCCTGGCCATCAGCCTGGGCTACTTCGGCCCCAAGTGCTACATGATCCTCTTCTACCCGGAGCGCAACACGCCCGCCTACTTCAACAGCATGATCCAGGGCTACACCATGAGGAGGGACTAG hT1R2 conceptual translation (SEQ ID NO 21)MGPRAKTICSLFFLLWVLAEPAENSDFYLPGDYLLGGLFSLHANMKGIVHLN (SEQ ID NO. 21)FLQVPMCKEYEVKVIGYNLMQAMRFAVEEINNDSSLLPGVLLGYEIVDVCYISNNVQPVLYFLAHEDNLLPIQEDYSNYISRVVAVIGPDNSESVMTVANFLSLFLLPQITYSAISDELRDKVRFPALLRTTPSADHHVEAMVQLMLHFRWNWIIVLVSSDTYGRDNGQLLGERVARRDICIAFQETLPTLQPNQNMTSEERQRLVTIVDKLQQSTARVVVVFSPDLTLYHFFNEVLRQNFTGAVWIASESWAIDPVLHNLTELGHLGTFLGITIQSVPIPGFSEFREWGPQAGPPPLSRTSQSYTCNQECDNCLNATLSFNTILRLSGERVVYSVYSAVYAVAHALHSLLGCDKSTCTKRWYPWQLLEEIWKVNFTLLDHQIFFDPQGDVALHLEIVQWQWDRSQNPFQSVASYYPLQRQLKNIQDISWHTVNNTIPMSMCSKRCQSGQKKKPVGIHVCCFECIDCLPGTFLNHTEDEYECQACPNNEWSYQSETSCFKRQLVFLEWHEAPTIAVALLAALGFLSTLAILVIFWRHFQTPIVRSAGGPMCFLMLTLLLVAYMWPVYVGPPKVSTCLCRQALFPLCFTICISCIAVRSFQIVCAFKMASRFPRAYSYWVRYQGPYVSMAFITVLKMVIWIGMLATGLSPTTRTDPDDPKITIVSCNPNYRNSLLFNTSLDLLLSWGFSFAYMGKELPTNYNEAKFITLSMTFYFTSSVSLCTFMSAYSGVLVTIVDLLVTVLNLLAISLGYFGPKCYMILFYPERNTPAYFNSMIQGYTM RRD

Example 7

Methods for Heterologous Expression of T1Rs in Heterologous Cells

An HEK-293 derivative (Chandrashekar et al., Cell 100(6): 703-11(2000)), which stably expresses Gal 5, was grown and maintained at 37°C. in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplementedwith 10% FBS, MEM non-essential amino acids (Gibco BRL), and 3 μg/mlblasticidin. For calcium-imaging experiments, cells were first seededonto 24-well tissue-culture plates (approximately 0.1 million cells perwell), and transfected by lipofection with Mirus Translt-293 (PanVera).To minimize glutamate-induced and glucose-induced desensitization,supplemented DMEM was replaced with low-glucose DMEM/GlutaMAX (GibcoBRL) approximately 24 hours after transfection. 24 hours later, cellswere loaded with the calcium dye Fluo-4 (Molecular Probes), 3 μM inDulbecco's PBS buffer (DPBS, GibcoBRL), for 1.5 hours at roomtemperature. After replacement with 250 μl DPBS, stimulation wasperformed at room temperature by addition of 200 μl DPBS supplementedwith taste stimuli. Calcium mobilization was monitored on a AxiovertS100 TV microscope (Zeiss) using Imaging Workbench 4.0 software (Axon).T1R1/T1R3 and T1R2/T1R3 responses were strikingly transient—calciumincreases rarely persisted longer than 15 seconds—and asynchronous. Thenumber of responding cells was thus relatively constant over time;therefore, cell responses were quantitated by manually counting thenumber of responding cells at a fixed time point, typically 30 secondsafter stimulus addition.

Example 8

Human T1R2/T1R3 Functions as a Sweet Taste Receptor

HEK cells stably expressing Gα15 were transiently transfected with humanT1R2, T1R3 and T1R2/T1R3, and assayed for increases in intracellularcalcium in response to increasing concentrations of sucrose (FIG. 1(a)). Also, T1R2/T1R3 dose responses were determined for several sweettaste stimuli (FIG. 1( b)). The maximal percentage of responding cellswas different for different sweeteners, ranging from 10-30%. Forclarity, dose responses were normalized to the maximal percentage ofresponding cells. The values in FIG. 1 represent the mean±s.e. of fourindependent responses. X-axis circles mark psychophysical detectionthresholds determined by taste testing. Gurmarin (50-full dilution of afiltered 10 g/l Gymnema sylvestre aqueous extract) inhibited theresponse of T1R2/T1R3 to 250 mM sucrose, but not the response ofendogenous β2-adrenergic receptor to 20 μM isoproterenol (FIG. 1( b)).FIG. 1( c) contains the normalized response of T1R2/T1R3 co-expressingcell lines to different sweeteners(sucrose, aspartame, tryptophan andsaccharin)

Example 9

Rat T1R2/T1R3 also Functions as a Sweet Taste Receptor

HEK cells stably expressing Gal were transiently transfected withhT1R2/hT1R3, rT1R2/rT1R3, hT1R2/rT1R3, and rT1R2/hT1R3. Thesetransfected cells were then assayed for increased intracellular calciumin response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and0.05 of monellin. The results with sucrose and aspartame are containedin FIG. 2 and indicate that rT1R2/rT1R3 also functions as a sweet tastereceptor. Also, these results suggest that T1R2 may control T1R2/T1R3ligand specificity.

Example 10

Human T1R1/T1R3 Functions as Umami Taste Receptors

HEK cells stably expressing Gα15 were transiently transfected with humanT1R1, T1R3 and T1R1/T1R3 and assayed for increases in intracellularcalcium in response to increasing concentrations of glutamate (FIG. 3(a) and 0.5 mM glutamate), 0.2 mM IMP, and 0.5 mM glutamate plus 0.2 mMIMP (FIG. 3( b)). Human T1R1/T1R3 dose responses were determined forglutamate in the presence and absence of 0.2 mM IMP (FIG. 3( c)). Themaximal percentages of responding cells was approximately 5% forglutamate and approximately 10% for glutamate plus IMP. For clarity,does responses are normalized to the maximal percentage of respondingcells. The values represent the mean±s.e. of four independent responses.X-axis circles mark taste detection thresholds determined by tastetesting.

Example 11

PDZIP as an Export Sequence

The six residue PDZIP sequence (SVSTW (SEQ ID NO:22)) was fused to theC-terminus of hT1R2 and the chimeric receptor (i.e. hT1R2-PDZIP) wastransfected into an HEK-293 host cell. The surface expression of hT1R2was then monitored using immunofluorescence and FACS scanning data. Asshown in FIGS. 6A and 6B, the inclusion of the PDZIP sequence increasedthe surface expression of hT1R2-PDZIP relative to hT1R2.

PKZIP Sequence

SVSTVV (SEQ ID NO:22)

More specifically, FIG. 4A shows an immunofluorescence staining ofmyc-tagged hT1R2 demonstrating that PDZIP significantly increases theamount of hT1R2 protein on the plasma membrane. FIG. 4B shows FACSanalysis data demonstrating the same result.—Cells expressing myc-taggedhT1R2 are indicated by the dotted line and cells expressing myc-taggedhT1R2-PDZIP are indicated by the solid line.

Example 12

Generation of Cell Lines that Stably Co-Express T1R1/T1R3 or T1R2/T1R3

Human cell lines that stably co-express human T1R2/T1R3 or humanT1R1/T1R3 were generated by transfecting linearized PEAK10-derived (EdgeBiosystems) vectors containing pCDNA 3.1/ZEO-derived (Invitrogen)vectors respectively containing hT1R1 or hT1R2 expression construct(plasmid SAV2485 for T1R1, SAV2486 for T1R2) and hT1R3 (plasmid SXV550for T1R3) into a G_(α15) expressing cell line. Specifically, T1R2/T1R3stable cell lines were produced by co-transfecting linearized SAV2486and SXV550 into Aurora Bioscience's HEK-293 cell line that stablyexpresses G_(α15). T1R1/T1R3 stable cell lines were produced byco-transfecting linearized SAV2485 and SXV550 into the same HEK-293 cellline that stably expresses Ga₁₅. Following SAV2485/SCV550 andSAV2486/SXV550 transfections, puromycin-resistant and zeocin-resistantcolonies were selected, expanded, and tested by calcium imaging forresponses to sweet or umami taste stimuli. Cells were selected in 0.0005mg/ml puromycin (CALBIOCHEM) and 0.1 mg/ml zeocin (Invitrogen) at 37° C.in low-glucose DMEM supplemented with GlutaMAX, 10% dialyzed FBS, and0.003 mg/ml blasticidin. Resistant colonies were expanded, and theirresponses to sweet taste stimuli evaluated by Fluorescence microscopy.For automated fluorimetric imaging on VIPR-II instrumentation (AuroraBiosciences), T1R2/T1R3 stable cells were first seeded onto 96-wellplates (approximately 15,000 cells per well). Twenty-four hours later,cells were loaded with the calcium dye fluo-3-AM (Molecular Probes),0.005 mM in PBS, for one hour at room temperature. After replacementwith 70 ml PBS, stimulation was performed at room temperature byaddition of 70 ml PBS supplemented with taste stimuli. Fluorescence (480nm excitation and 535 nm emission) responses from 20 to 30 secondsfollowing compound addition were averaged, corrected for backgroundfluorescence measured prior to compound addition, and normalized to theresponse to 0.001 mM ionomycin (CALBIOCHEM), a calcium ionophore.

It was then observed that when these cell lines were contacted withsweet or umami, that for active clones typically 80-100% of cellsresponded to taste stimuli. Unexpectedly, the magnitude of individualcell responses was markedly larger than that of transiently transfectedcells.

Based on this observation, the inventors tested the activity of T1Rstable cell lines by automated fluorescence imaging using AuroraBioscience's VIPR instrumentation as described above. The responses oftwo T1R1/T1R3 and one T1R2/T1R3 cell line are shown in FIG. 5 and FIG. 6respectively.

Remarkably, the combination of increased numbers of responding cells andincreased response magnitudes resulted in a greater than 10-foldincrease in activity relative to transiently transfected cells. (By wayof comparison, the percent ionomycin response for cells transientlytransfected with T1R2/T1R3 was approximately 5% under optimalconditions.) Moreover, dose responses obtained for stably expressedhuman T1R2/T1R3 and T1R1/T1R3 correlated with human taste detectionthresholds. The robust T1R activity of these stable cell lines suggeststhat they are well suited for use in high-throughput screening ofchemical libraries in order to identify compounds, e.g. small molecules,that modulate the sweet or umami taste receptor and which thereforemodulate, enhance, block or mimic sweet or umami taste.

While the foregoing detailed description has described severalembodiments of the present invention, it is to be understood that theabove description is illustrative only and not limiting of the disclosedinvention. The invention is to be limited only by the claims whichfollow.

1. An in vitro method for identifying a compound that putatively elicitsor modulates taste in a human subject based on its effect on theactivation of a taste receptor comprising a human T1R2 polypeptidecomprising: (i) screening one or more compounds in a functional assaythat detects compounds which activate or modulate (enhance or inhibit)the activation of a taste receptor comprising a human T1R2 polypeptideselected from the group consisting of: (a) a human T1R2 polypeptidehaving the amino acid sequence in SEQ. ID. NO: 21; (b) a human T1R2polypeptide that possesses at least 90% sequence identity to thepolypeptide in SEQ. ID. NO: 21; (c) a human T1R2 polypeptide whichencoded by a nucleic acid sequence that hybridizes to the T1R2polypeptide coding region of the nucleic acid sequence in SEQ. ID. NO:23 under stringent hybridization conditions which are incubation in 50%formamide, 5X SSC and 0.1% SDS, with wash in 0.2X SSC and 0.1% SDS at 65degrees C and which taste receptor comprising said human T1R2polypeptide specifically binds to a ligand that also specifically bindsto the human T1R2 polypeptide in SEQ ID NO:21; (ii) identifyingcompounds (i) that putatively elicit or modulate T1R2polypeptide-associated taste subject based on their (a) activation ormodulation (inhibition or enhancement) of the activation of said T1R2polypeptide according to (a), (b), or (c), in said functional assay (i).2. The method of claim 1, wherein said T1R2 polypeptide has the aminoacid sequence in SEQ. ID. NO:
 21. 3. The method of claim 1, wherein saidT1R2 polypeptide has an amino acid sequence that possesses at least 90%sequence identity to the polypeptide in SEQ. ID. NO:
 21. 4. The methodof claim 1, wherein said T1R2 polypeptide has an amino acid sequencethat possesses at least 95% sequence identity to the polypeptide in SEQ.ID. NO:
 21. 5. The method of claim 1, wherein said T1R2 polypeptide hasan amino acid sequence that possesses at least 96% sequence identity tothe polypeptide in SEQ. ID. NO:
 21. 6. The method of claim 1, whereinthe T1R2 polypeptide possesses at least 97% sequence identity to thepolypeptide in SEQ. ID. NO:
 21. 7. The method of claim 1, wherein saidT1R2 polypeptide has an amino acid sequence that possesses at least 97%sequence identity to the polypeptide in SEQ. ID. NO:
 21. 8. The methodof claim 1, wherein said T1R2 polypeptide has an amino acid sequencethat possesses at least 98% sequence identity to the polypeptide in SEQ.ID. NO:
 21. 9. The method of claim 1, wherein said T1R2 polypeptide hasan amino acid sequence that possesses at least 99% sequence identity tothe polypeptide in SEQ. ID. NO:
 21. 10. The method of claim 1, whereinsaid T1R2 polypeptide is encoded by a nucleic acid sequence thathybridizes to the T1R2 coding region in SEQ. ID. NO: 23 under stringenthybridization conditions.
 11. The method of claim 1, wherein said T1R2polypeptide is expressed in a cell.
 12. The method of claim 11, whereinsaid cell is intact or permeabilized.
 13. he method of claim 1, whereinsaid T1R2 polypeptide is comprised in a membrane extract.
 14. The methodof claim 11, wherein said T1R2 polypeptide is expressed on the surfaceof said cell.
 15. The method of claim 11, wherein the cell is aeukaryotic cell.
 16. The method of claim 15, wherein said cell is ayeast, insect, amphibian or mammalian cell.
 17. The method of claim 15,wherein the cell is a CRO, HEK-293, COS or Xenopus oocyte.
 18. Themethod of claim 11, wherein said cell expresses a G protein.
 19. Themethod of claim 18, wherein said G protein is G_(α15) or G_(α16) orgustducin.
 20. The method of claim 1, wherein said functional assaydetects the effect of said compound on phosphorylation of the T1R2polypeptide.
 21. The method of claim 1, wherein the functional assaydetects the effect of said compound on the dissociation of said T1R2polypeptide and a G protein.
 22. The method of claim 1, wherein thefunctional assay detects the effect of said compound on arrestintranslocation.
 23. The method of claim 1, wherein the functional assaydetects the effect of said compound on second messenger(s).
 24. Themethod of claim 1, wherein the functional assay detects the effect ofsaid compound on signal transduction.
 25. The method of claim 1, whereinthe functional assay is a fluorescent polarization assay.
 26. The methodof claim 24, wherein said functional assay is a GTPγ³⁵S assay.
 27. Themethod of claim 24, wherein said functional assay detects changes incAMP, cGMP or IP3.
 28. The method of claims 1, wherein said functionalassay detects changes in intracellular calcium.
 29. The method of claim28, which uses a calcium-sensitive dye.
 30. The method of claim 1 whichdetects the effect of said compound on G protein activation by said T1R2polypeptide.
 31. The method of claim 30, wherein said G protein isG_(α15), G_(α16) or gustducin.
 32. The method of claim 1, wherein saidT1R2 polypeptide in said functional assay is stably or transientlyexpressed by a cell.
 33. The method of claim 1, wherein the functionalassay detects changes in ionic polarization of a cell or membrane thatexpresses the T1R2 polypeptide.
 34. The method of claim 33, wherein ionpolarization is detected by a voltage-clamp or patch-clamp method. 35.The method of claim 1, wherein said functional assay comprises aradiolabeled ion flux assay or fluorescence assay that detects T1R2activity using a voltage-sensitive dye.
 36. The method if claim 1,wherein said assay comprises a fluorescent polarization or FRET assay.37. The method of claim 1, wherein said assay detects changes inadenylate cyclase activity.
 38. The method of claim 1 wherein thefunctional assay detects changes in ligand-dependent coupling of saidT1R2 polypeptide with a G protein.
 39. The method of claim 38 whereinsaid G protein is G_(α15) or G_(α16) or gustducin.
 40. The method ofclaim 1, wherein said functional assay detects changes in intracellularcAMP or cGMP.
 41. The method of claim 1, wherein said assay measures theeffect of said compound on transmitter or hormone release.
 42. Themethod of claim 1 wherein said functional assay detects the effect ofsaid compound on the transcription of a gene of interest.
 43. The methodof claim 42, wherein said gene is a reporter selected fromchioramphenicol acetyltransferase, luciferase, 3′-galactosidase andalkaline phosphatase.
 44. The method of claim 1, wherein the functionalassay is a high throughput assay.
 45. The method of 44, wherein saidfunctional assay screens a library of compounds.
 46. The method of claim45 wherein said library is a combinatorial chemical library.
 47. Themethod of claim 45 wherein said library comprises at least 1000compounds.
 48. The method of claim 1, wherein the effect of saidputative T1R2 taste modulator is assayed in vivo for its effect on T1R2receptor polypeptide-associated taste.
 49. The method of claim 48 whichis used to assay the effect of said compound on the taste of aparticular compound.
 50. The method of claim 49, wherein said assay isused to detect the effect of said compound on sweet or umami taste. 51.The method of claim 1 wherein said T1R2 polypeptide is expressed by ataste cell, gastroepithelial cell, geschmackstreifen, oral cavity, orepiglotttis cell.
 52. The method of claim 1 wherein said T1R2 polpeptideis a expressed by a gastroepithelial cell, esophageal cell, stomach cellderived from the plate.
 53. The method of claim 51 wherein the tastecell is a foliate, fungiform, or circumvallate cell.